The relevance of the superior cervical ganglion for cardiac autonomic innervation in health and disease: a systematic review.

PURPOSE: The heart receives cervical and thoracic sympathetic contributions. Although the stellate ganglion is considered the main contributor to cardiac sympathetic innervation, the superior cervical ganglia (SCG) is used in many experimental studies. The clinical relevance of the SCG to cardiac innervation is controversial. We investigated current morphological and functional evidence as well as controversies on the contribution of the SCG to cardiac innervation. METHODS: A systematic literature review was conducted in PubMed, Embase, Web of Science, and COCHRANE Library. Included studies received a full/text review and quality appraisal. RESULTS: Seventy-six eligible studies performed between 1976 and 2023 were identified. In all species studied, morphological evidence of direct or indirect SCG contribution to cardiac innervation was found, but its contribution was limited. Morphologically, SCG sidedness may be relevant. There is indirect functional evidence that the SCG contributes to cardiac innervation as shown by its involvement in sympathetic overdrive reactions in cardiac disease states. A direct functional contribution was not found. Functional data on SCG sidedness was largely unavailable. Information about sex differences and pre- and postnatal differences was lacking. CONCLUSION: Current literature mainly supports an indirect involvement of the SCG in cardiac innervation, via other structures and plexuses or via sympathetic overdrive in response to cardiac diseases. Morphological evidence of a direct involvement was found, but its contribution seems limited. The relevance of SCG sidedness, sex, and developmental stage in health and disease remains unclear and warrants further exploration.

Quantification of the intrinsic neural plexus of the heart - The missing link in histological tissue analysis.

BACKGROUND AND OBJECTIVE: The heart is under strict regulation of the autonomic nervous system, during which, in a healthy state, the effects of sympathetic and parasympathetic branches are balanced. In recent years, there has been increasing interest in pathological remodeling and outgrowth of cardiac autonomic nerves in relation to arrhythmogenesis. However, the small size of the cardiac nerves in relatively large tissues renders research using histological quantification of these nerves extremely challenging and usually relies on quantification of the nerve density in selected regions of interest only. Our aim was to develop a method to be able to quantify the histological nerve density in transmural tissue sections. METHODS: Here we describe a novel workflow that enables visualization and quantification of variable innervation types and their heterogeneity within transmural myocardial tissue sections. A custom semiautomatic workflow for the quantification of cardiac nerves involving Python, MATLAB and ImageJ is provided and described in this protocol in a stepwise and detailed manner. REPRESENTATIVE RESULTS: The results of two example tissue sections are represented in this paper. An example tissue section taken from the infarction core with a high heterogeneity value of 0.20, 63.3% normal innervation, 12.2% hyperinnervation, 3.6% hypoinnervation and 21.0% denervation. The second example tissue section taken from an area of the left ventricle remote from the infarction showed a low heterogeneity value of 0.02, 95.3% normal innervation, 3.8% hyperinnervation, 0.5% hypoinnervation and 0.5% denervation. CONCLUSIONS: This approach has the potential to be broadly applied to any research involving high-resolution imaging of nerves in large tissues.

Cardiac sympathetic hyperinnervation after myocardial infarction: a systematic review and qualitative analysis.

BACKGROUND: Cardiac sympathetic hyperinnervation after myocardial infarction (MI) is associated with arrhythmogenesis and sudden cardiac death. The characteristics of cardiac sympathetic hyperinnervation remain underexposed. OBJECTIVE: To provide a systematic review on cardiac sympathetic hyperinnervation after MI, taking into account: (1) definition, experimental model and quantification method and (2) location, amount and timing, in order to obtain an overview of current knowledge and to expose gaps in literature. METHODS: References on cardiac sympathetic hyperinnervation were screened for inclusion. The included studies received a full-text review and quality appraisal. Relevant data on hyperinnervation were collected and qualitatively analysed. RESULTS: Our literature search identified 60 eligible studies performed between 2000 and 2022. Cardiac hyperinnervation is generally defined as an increased sympathetic nerve density or increased number of nerves compared to another control group (100%). Studies were performed in a multitude of experimental models, but most commonly in male rats with permanent left anterior descending (LAD) artery ligation (male: 63%, rat: 68%, permanent ligation: 93%, LAD: 97%). Hyperinnervation seems to occur mainly in the borderzone. Quantification after MI was performed in regions of interest in µm2/mm2 (41%) or in percentage of nerve fibres (46%) and the reported amount showed a great variation ranging from 439 to 126,718 µm2/mm2. Hyperinnervation seems to start from three days onwards to >3 months without an evident peak, although studies on structural evaluation over time and in the chronic phase were scarce. CONCLUSIONS: Cardiac sympathetic hyperinnervation after MI occurs mainly in the borderzone from three days onwards and remains present at later timepoints, for at least 3 months. It is most commonly studied in male rats with permanent LAD ligation. The amount of hyperinnervation differs greatly between studies, possibly due to differential quantification methods. Further studies are required that evaluate cardiac sympathetic hyperinnervation over time and in the chronic phase, in transmural sections, in the female sex, and in MI with reperfusion.

KEY MESSAGESCardiac sympathetic hyperinnervation occurs three days after MI mainly in the borderzone and remains present at all timepoints.It is most commonly studied in male rats with permanent LAD ligation.The amount of hyperinnervation differs greatly between studies, possibly due to the differential quantification methods.

Quantification of Large Transmural Biopsies Reveals Heterogeneity in Innervation Patterns in Chronic Myocardial Infarction.

BACKGROUND: Abnormal cardiac innervation plays an important role in arrhythmogenicity after myocardial infarction (MI). Data regarding reperfusion models and innervation abnormalities in the medium to long term after MI are sparse. Histologic quantification of the small-sized cardiac nerves is challenging, and transmural analysis has not been performed. OBJECTIVES: This study sought to assess cardiac innervation patterns in transmural biopsy sections in a porcine reperfusion model of MI (MI-R) using a novel method for nerve quantification. METHODS: Transmural biopsy sections from 4 swine (n = 83) at 3 months after MI-R and 3 controls (n = 38) were stained with picrosirius red (fibrosis) and beta-III-tubulin (autonomic nerves). Biopsy sections were classified as infarct core, border zone, or remote zone. Each biopsy section was analyzed with a custom software pipeline, allowing calculation of nerve density and classification into innervation types at the 1 × 1-mm resolution level. Relocation of the classified squares to the original biopsy position enabled transmural quantification and innervation heterogeneity assessment. RESULTS: Coexisting hyperinnervation, hypoinnervation, and denervation were present in all transmural MI-R biopsy sections. The innervation heterogeneity was greatest in the infarct core (median: 0.14; IQR: 0.12-0.15), followed by the border zone (median: 0.05; IQR: 0.04-0.07; P = 0.02) and remote zone (median: 0.02; IQR: 0.02-0.03; P < 0.0001). Only in the border zone was a positive linear relation between fibrosis and innervation heterogeneity observed (R = 0.79; P < 0.0001). CONCLUSIONS: This novel method allows quantification of nerve density and heterogeneity in large transmural biopsy sections. In the chronic phase after MI-R, alternating innervation patterns were identified within the same biopsy section. Persistent innervation heterogeneity, in particular in the border zone biopsy sections, may contribute to late arrhythmogenicity.

Volume-Weighted Unipolar Voltage Predicts Heart Failure Mortality in Patients With Dilated Cardiomyopathy and Ventricular Arrhythmias.

BACKGROUND: Patients with dilated cardiomyopathy (DCM) who are undergoing catheter ablation of ventricular arrhythmias (VAs) are at risk of rapidly progressive heart failure (HF). Endocardial voltages decrease with loss of viable myocardium. Global left ventricular (LV) voltage as a surrogate for the amount of remaining viable myocardium may predict prognosis. OBJECTIVES: This study evaluated whether the newly proposed parameter volume-weighted (vw) unipolar voltage (UV) can predict HF-related adverse outcomes (HFOs), including death, heart transplantation, or ventricular assist device implantation, in DCM. METHODS: In consecutive patients with DCM referred for VA ablation, vwUV was calculated by mathematically integrating UV over the left ventricle, divided by the endocardial LV surface area and wall thickness. Patients were followed for HFOs. RESULTS: A total of 103 patients (57 ± 14 years of age; left ventricular ejection fraction [LVEF], 39% ± 13%) were included. Median vwUV was 9.75 (IQR: 7.27-12.29). During a median follow-up of 24 months (IQR: 8-47 months), 25 patients (24%) died, and 16 had HFOs 7 months (IQR: 1-18 months) after ablation. Patients with HFOs had significantly lower LVEF (29% ± 10% vs 41% ± 12%), vw bipolar voltage (BV) (3.00 [IQR: 2.47-3.53] vs 5.00 [IQR: 4.12-5.73]), and vwUV (5.94 [IQR: 5.28-6.55] vs 10.37 [IQR: 8.82-12.81]; all P < 0.001), than patients without HFOs. In Cox regression analysis and goodness-of-fit tests, vwUV was the strongest and independent predictor for HFOs (HR: 3.68; CI: 2.09-6.45; likelihood ratio chi-square, 33.05; P < 0.001). CONCLUSIONS: The novel parameter vwUV, as a surrogate for the amount of viable myocardium, identifies patients with DCM with VA who are at high risk for HF progression and mortality.

Ventricular Arrhythmia Substrate Distribution and Its Relation to Sympathetic Innervation in Nonischemic Cardiomyopathy Patients.

BACKGROUND: Nonischemic cardiomyopathy patients referred for catheter ablation of ventricular arrhythmias (VAs) typically have either inferolateral (ILS) or anteroseptal (ASS) VA substrate locations, with poorer outcomes for ASS. Sympathetic denervation is an important determinant of arrhythmogenicity. Its relation to nonischemic fibrosis in general and to the different VA substrates is unknown. OBJECTIVES: This study sought to evaluate the association between VA substrates, myocardial fibrosis, and sympathetic denervation. METHODS: Thirty-five patients from the Leiden Nonischemic Cardiomyopathy Study, who underwent electroanatomic voltage mapping and iodine-123 metaiodobenzylguanidine imaging between 2011 and 2018 were included. Late gadolinium-enhanced cardiac magnetic resonance data were collected when available. The relation between global cardiac sympathetic innervation and area-weighted unipolar voltage (UV) as a surrogate for diffuse fibrosis was evaluated. For regional analysis, patients were categorized as ASS or ILS. The distribution of low UV, sympathetic denervation, and late gadolinium enhancement (LGE) scar were compared using the 17-segment model. RESULTS: Median area-weighted UV was 12.3 mV in patients with normal sympathetic innervation and 8.7 mV in patients with sympathetic denervation. Global sympathetic denervation correlated with diffuse myocardial fibrosis (R = 0.53; P = 0.02). ILS (n = 13) matched with low UV, sympathetic denervation, and LGE scar in all patients, whereas ASS (n = 11) matched with low UV in all patients, with LGE scar in 63% (P = 0.20), but with sympathetic denervation in only 27% of patients (P = 0.0002). CONCLUSIONS: Global cardiac sympathetic denervation is related to fibrosis in nonischemic cardiomyopathy patients with VA. The mismatch between regional fibrosis and preserved innervation for ASS may contribute to a VA substrate difficult to control by catheter ablation.

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing.

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome. Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice. This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.