Disruption of afferent neural circuits leads to arrhythmia in the animal model of hereditary sensory and autonomic neuropathy 6

Abstract

Hereditary sensory and autonomic neuropathies (HSANs) are a group of recessive genetic disorders affecting the sensory and autonomic components of the peripheral nervous system (PNS). Compared with somatosensory dysfunctions, the pathogenesis of visceral dysfunction in HSANs remains understudied. This study investigated the neural circuit mechanisms underlying the arrhythmias observed in conditional Dystonin (Dst) gene-trap mice, an animal model of HSAN type VI (HSAN-VI) in which Cre recombinase inactivates Dst expression in selective neural circuits. Inactivation of the Dst gene in PNS neurons using Advillin-Cre caused the degeneration of sensory and sympathetic ganglionic neurons. This was accompanied by arrhythmia, characterized by increased heart rate variability and irregular pulse frequency, which was prominent under isoflurane anesthesia and occurred in the absence of protein aggregate cardiomyopathy. Furthermore, selective inactivation of the Dst gene in PNS sensory neurons using Vglut2-Cre resulted in similar dysregulation of cardiac rhythm. These findings suggest that arrhythmias caused by Dst mutations arise from the disruption of visceral afferent circuits, and that these neural circuits could be potential therapeutic targets for visceral dysfunction in HSAN-VI.

1. Introduction

Hereditary sensory and autonomic neuropathies (HSANs) comprise a group of clinically and genetically heterogeneous neurodegenerative disorders that affect the peripheral nervous system (PNS) (Rotthier et al., 2012). Patients with HSANs exhibit neurodegeneration in both the sensory and autonomic nervous systems. Because HSANs disrupt a wide range of neural networks, identifying the specific circuits responsible for individual symptoms and those amenable to targeted therapeutic intervention remains a major challenge. HSAN type VI (HSAN-VI) is caused by loss-of-function mutations in the Dystonin (DST) gene (Edvardson et al., 2012). In mice, naturally occurring dystonia musculorum (dt) mutation results in sensory neuron degeneration and abnormal motor behaviors, including ataxia and dystonia (Duchen et al., 1964; Horie et al., 2016). Since the identification of Dst as the causative gene for the dt phenotype (Brown et al., 1995; Guo et al., 1995), dt mice have been widely used to investigate the pathogenic mechanisms of HSAN-VI and to explore potential therapeutic strategies (Ferrier et al., 2014). To dissect the role of Dst in specific neural circuits, we previously generated a multipurpose Dst allele that enables cell type- or neural circuit-selective inactivation and restoration via Flip-excision (FLEX) technology (Schnütgen et al., 2005; Horie et al., 2014; Hossain et al., 2018). Using this genetic tool, we demonstrated that degeneration of proprioceptive neurons in the dorsal root ganglia (DRG) is causative for the movement disorders observed in dt mice (Horie et al., 2020; Yoshioka et al., 2024).

In addition to somatosensory dysfunction, patients with HSAN-VI exhibit various visceral abnormalities, including impaired cardiovascular reflexes, sexual dysfunction, pupillary abnormalities, and gastrointestinal dysmotility (Manganelli et al., 2017). However, the underlying neural circuit mechanisms remain poorly understood. Reduced gastrointestinal motility in dt mice has been suggested to be associated with degeneration of the vagus nerve (Lynch-Godrei et al., 2020). The vagus nerve comprises parasympathetic efferent fibers as well as visceral afferent fibers (Waise et al., 2018). Notably, signs of neurodegeneration have been reported in the vagal ganglia, which include visceral sensory neurons, in dt mice (Ichikawa et al., 2006; Lynch-Godrei et al., 2020). These observations raise the possibility that disruption of visceral afferent circuits contributes to visceral dysfunctions in HSAN-VI.

The Dst gene encodes tissue-specific isoforms, including DST-a, DST-b, and DST-e, which are predominantly expressed in neural, muscular, and cutaneous tissues, respectively (Künzli et al., 2016; Horie et al., 2017; Yoshioka, 2024). These major DST isoforms are thought to play essential roles in maintaining the integrity of their respective tissues by acting as cytoskeletal linker proteins. We previously reported that isoform-specific Dst-b mutant mice develop late-onset protein aggregate-associated cardiomyopathy and arrhythmia, suggesting that cell-autonomous defects in cardiomyocytes can lead to dysregulation of cardiac function (Yoshioka et al., 2022). This observation was subsequently supported by a clinical report identifying DST-b-specific mutations as a cause of congenital myopathy and cardiomyopathy in human patients (Jacob et al., 2025). Nevertheless, it remains unclear whether the cardiovascular dysregulation observed in patients with HSAN-VI arises primarily from abnormalities in neural circuits or from intrinsic defects in cardiomyocytes. To address this issue, we performed conditional inactivation of the Dst gene in sensory and/or autonomic nervous systems to elucidate the respective contributions of PNS neural circuits to cardiac rhythm regulation in HSAN-VI.

2. Materials and methods

2.1. Animals

We used Dst^Gt(E182H05) mice (Dst^Gt; MGI: 3917429, Horie et al., 2014) derived from the ES clone obtained from GGTC (ID: E182H05), and Dst-b^E2610Ter mice (Dst^em1Htak, MGI:7423674, Yoshioka et al., 2022). Dst^Gt mice were crossed with several Cre-driver mice including PNS neuron-selective Advillin (Avil)-Cre mice (Avil^tm2(cre)Fawa; MGI:4459942, Hasegawa et al., 2007; Zhou et al., 2010), and glutaminergic neuron-selective Vglut2-Cre mice (Slc17a6^tm2(cre)Lowl/J; MGI: 5300532, Vong et al., 2011). Ai14 reporter mice (B6. Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J; MGI:3813512, Madisen et al., 2010) were crossed with Avil-Cre mice or Vglut2-Cre mice. The functional Dst^Gt-inv allele was inverted from Dst^Gt allele by FLP recombinase, and mutant Dst^Gt-DO allele was inverted from Dst^Gt-inv allele by Cre recombinase (Horie et al., 2014). Mutant mice of Dst^Gt, Dst^Gt-inv, and Dst^Gt-DO were maintained in each line. To perform Cre-mediated conditional inactivation of the Dst expression, gene-trap mice harboring multipurpose Dst alleles were crossed with each Cre driver mouse. The animal experiments were approved by the Internal Review Board of Niigata University. Mice were maintained at 23 ± 3 °C, 50 ± 10% humidity, and 12 h light/dark cycle with food and water available ad libitum.

2.2. Tissue preparations and in situ hybridization

In situ hybridization was carried out as described in previous studies (Yoshioka et al., 2020, 2022, 2024). For tissue preparation, mice were euthanized by intraperitoneal injection of pentobarbital sodium (100 mg/kg body weight) and transcardially perfused with 0.01 M phosphate-buffered saline (PBS), followed by ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4. Dissected tissues were immersed in the same fixative overnight. Neural tissues were cryoprotected by immersion in 20% sucrose in 20 mM PBS (pH 7.4) until they sank, frozen in liquid nitrogen, embedded in Tissue-Tek OCT compound (Sakura Finetek Japan, Tokyo, Japan), and sectioned at 16 μm using a cryostat (Leica CM1850 UV; Leica, Wetzlar, Germany; HM525 NX; Thermo Fisher Scientific, Waltham, MA). Heart tissues were dehydrated through a graded ethanol series followed by xylene and embedded in paraffin (Paraplast Plus^®, P3683; Sigma-Aldrich, St. Louis, MO, USA). Consecutive 10-μm-thick paraffin sections were cut using a rotary microtome (HM325; Thermo Fisher Scientific), mounted on MAS-coated glass slides (Matsunami Glass, Osaka, Japan), and air-dried overnight on a hot plate at 37 °C. For In situ hybridization the following probes were used: mouse Dst-plakin (Genbank accession number, NM_001276764, nt 2185–3396), and mouse Nppa, also known as atrial natriuretic peptide (ANP, GenBank accession, BC089615, nt 124–529).

2.3. Immunohistochemistry

Immunohistochemical analyses were performed on paraffin sections as described in previous studies (Yoshioka et al., 2020, 2022, 2024). Deparaffinized sections were subjected to microwave irradiation in 10 mM citric acid buffer, pH 6.0 for 5 min, or frozen sections were used without antigen retrieval. Sections were incubated overnight at 4 °C with following primary antibodies: rabbit polyclonal anti-PGP9.5 antibody (1:1000; ENZO Life Sciences, Farmingdale; ADI-905-520-1), rabbit polyclonal anti-TH antibody (1;500; Pel-Freez Biologicals, Rogers, AR; P40101), rabbit polyclonal anti-ATF3 antibody (1:2000; Santa Cruz Biotechnology, Dallas, TX; sc-188), rabbit polyclonal anti-Sprr1a antibody (1:1000; ABclonal Technology, Woburn, MA; A17535), rabbit polyclonal anti- HspB1/HSP25 antibody (1:800; ABclonal Technology; A11156), rabbit polyclonal anti-HSPA1L antibody (1:500; ABclonal Technology; A1856), rabbit polyclonal anti-VAchT antibody (1:500; GeneTex, Irvine, CA; GTX133251), and mouse monoclonal anti-p62 antibody (1:200; BioLegend, San Diego, CA; MMS-5034) diluted in 0.1 M PBS with 0.01% triton X-100 (PBST) containing 0.5% skim milk. Sections were then incubated with horseradish peroxidase-conjugated secondary antibody (1:200; MBL, Nagoya, Japan) diluted in PBST containing 0.5% skim milk for 60 min at 37 °C. Between each step, sections were rinsed in PBST for 15 min. After rinsing sections in PBST, immunoreaction was visualized in 50 mM Tris buffer (pH 7.4) containing 0.01% diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide at 37 °C for 5 min. Sections were dehydrated through ethanol-xylene and coverslipped with Bioleit (23–1002; Okenshoji, Tokyo, Japan). For immunofluorescent staining, sections were incubated in mixtures of Alexa488- or Alexa594-conjugated antibodies (1:200; Invitrogen, CA) for 60 min at 37 °C. Mounted sections were air-dried and coverslipped. Digital images were taken with a microscope (BX53; Olympus, Tokyo, Japan) equipped with a digital camera (DP74, Olympus) and a confocal laser scanning microscopy (FV-1200, Olympus). TIF files were processed with Photoshop software (Adobe, San Jose, USA).

2.4. Measurements of electrocardiogram signal and quantification

Electrocardiogram (ECG) was recorded as described in previous study under anesthesia (Yoshioka et al., 2022). Mice were exposed to 3.5% isoflurane (Pfizer Inc., NY) for induction of anesthesia and then switched to 1.5% isoflurane for maintenance, during which ECG was recorded for a duration of 5 min. Three needle electrodes were inserted into the right and left forelimbs for recording, and right hindlimb for grounding. The ECG signals were amplified using AC amplifier (band pass: 0.1–1 kHz), and the signals were digitized with A/D converter (Power 1401, Cambridge Electronic Design Ltd., Cambridge, UK), at sampling rate of 2 kHz. The recorded ECG signals were analyzed using LabChart Pro software (version 8.1.30; ADInstruments, Bella Vista, Australia) with the ECG Analysis Module. Segments containing significant noise or motion artifacts were identified by visual inspection and excluded from the analysis to ensure data quality. Three epochs were defined for analysis: (i) the induction period under 3.5% isoflurane, (ii) the immediate post-transition period to 1.5% isoflurane, and (iii) the stabilized maintenance period 5 min post-transition. Within each epoch, the heart rate (HR), RR interval, and the standard deviation (SD) of RR intervals were calculated as indices of heart rate variability (HRV). To analyze ECG waveforms, at least 30 consecutive beats were averaged to determine the following parameters: the PR (PQ) interval (from the onset of the P wave to the start of the QRS complex), the QRS duration (from the start of the Q wave to the end of the S wave), and the QT interval (from the beginning of the QRS complex to the end of the T wave).

2.5. ECG recording in freely moving mice

To evaluate cardiac activity in unanesthetized mice, we performed chronic ECG recordings using a custom-built system in freely moving mice. Mice were anesthetized with 3.5% isoflurane. Following a midline skin incision, enamel-coated copper wires were implanted and positioned to sandwich the heart. This configuration was chosen to ensure optimal signal detection of the cardiac vector, thereby maximizing the R-wave amplitude and signal-to-noise ratio. The electrode leads were routed subcutaneously to the dorsal region and exteriorized. A stainless steel screw electrode was implanted into the occipital bone to serve as the system ground. Subsequently, all leads were soldered to a custom-made head-mounted connector. To ensure long-term stability, the connector was rigidly secured to the bone using dental resin (Super-Bond; Sun Medical, Shiga, Japan). Finally, the skin incisions were carefully sutured to facilitate post-operative recovery. Following a recovery period, ECG signals were recorded from free-moving mice. To initiate recording, the head-mounted connector was linked to the data acquisition system using a custom-made recording system (Unique Medical, Tokyo, Japan). This system, which extended from the head-mounted connector through an electrical swivel (slip-ring) to the amplifier, allowed for unrestricted movement within the observation cage. This flexible setup enabled stable, long-term data acquisition in a freely moving state without movement-induced stress. The ECG signals were amplified using AC amplifier (band- pass filter: 0.1–1 kHz), and the signals were digitized with A/D converter (Power 1401, Cambridge Electronic Design Ltd.), at sampling rate of 2 kHz. The recorded ECG signals were analyzed using LabChart Pro software (version 8.1.30; ADInstruments) with the ECG Analysis Module. Following the connection of the recording cable to head-connector, mice were allowed to habituate for 30 min before data collection started. For analysis, a 1-min segment was extracted during periods characterized by a stable baseline and minimal waveform variability. Segments containing significant noise or motion artifacts were identified by visual inspection and excluded from the analysis. To analyze ECG waveforms, at least 30 consecutive beats were averaged.

2.6. Tissue clearing protocol and imaging

The brain and spinal cord were made transparent for imaging using clear unobstructed brain/body imaging cocktails and computational analysis (CUBIC) protocol (Tainaka et al., 2018), as described previously (Yoshioka et al., 2024). The brains and spinal cords were dissected from mice following perfusion with PBS, pH 7.4, and 4% PFA in 0.1 M PB. The tissues were postfixed overnight in same fixative at 4 °C and washed with PBS. The samples were immersed in CUBIC-L [10% polyethylene glycol mono-p-isooctyphenyl ether (12969–25, Nacalai Tesque, Kyoto) and 10% N-butyldiethanolamine (B0725, Tokyo Chemical Industry, Tokyo) in water] with shake at 37 °C for 5 days. CUBIC-L was exchanged at 2 days after the immersion. The samples were then washed three times with PBS at room temperature for 2 h and then immersed in 50% CUBIC-R [45% 2,3-dimethyl-1-phenyl-5-pyrazolone (D1876, Tokyo Chemical Industry) and 30% nicotinamide (N0078, Tokyo Chemical Industry), pH adjusted to approximately 8 to 9 with N-butyldiethanolamine (B0725, Tokyo Chemical Industry, Tokyo) in water] diluted in water at room temperature for 5 h and then gently shaken in CUBIC-R at room temperature overnight. The samples were immersed in new CUBIC-R and kept until the microscopic observation. 3D fluorescent images were acquired with light sheet fluorescence microscopes (MVX10-LS, Olympus).

2.7. Statistical analysis

Statistical analysis was performed using t-test (Control vs. cGT) or two-way repeated measures analysis of variance (ANOVA) with genotype (Control vs. cGT) and epoch (i, ii, and iii) as the main factors. Post-hoc comparisons were conducted by Tukey to identify specific differences between groups. These analyses were carried out using the Easy R (EZR, Saitama Medical Center, Jichi Medical University, Japan, Kanda, 2013). Sample size (n) is the number of animals in each genotype.

3. Results

3.1. PNS neuron-selective inactivation of Dst gene leads to neurodegeneration

Previously, we generated multipurpose gene-trap mice carrying the Dst^Gt allele, which enables both Cre-mediated inactivation and restoration of Dst (Horie et al., 2014; Yoshioka et al., 2024). To achieve inactivation of Dst gene expression in PNS neurons, we used Avil-Cre mice, in which Cre-mediated recombination occurs in sensory neurons and sympathetic ganglionic neurons (Zurborg et al., 2011; Hunter et al., 2018). To confirm PNS neuron-selective recombination, Avil-Cre mice were crossed with Ai14 reporter mice, in which tdTomato expression is induced by Cre-mediated recombination (Figure 1). In Avil-Cre; Ai14 mice, tdTomato was observed in sensory neurons of the dorsal root ganglia (DRG) (Figure 1A) and vagal ganglia (Figure 1B), as well as in sympathetic ganglionic neurons of the superior cervical ganglion (SCG) (Figure 1C). These results indicate efficient Cre-mediated recombination in both PNS afferent and efferent neurons by the Avil-Cre mice.

Figure 1.

Cre-mediated reporter expression in sensory and autonomic ganglia of Advillin-Cre; Ai14 and Vglut2 − Cre; Ai14 mice. (A–C) The distribution of tdTomato fluorescent (red) was examined in the dorsal root ganglia (DRG), vagal ganglia, and superior cervical ganglia (SCG) of Advillin (Avil)-Cre; Ai14 and Vglut2-Cre; Ai14 mice to compare Cre-recombinase activity driven by the Avil and Vglut2 promoters. The Ai14 reporter allele expresses tdTomato following Cre-mediated recombination. (A) In the DRG of both Avil-Cre; Ai14 and Vglut2-Cre; Ai14 mice, tdTomato is expressed by many sensory neurons positive for PGP9.5. (B) In the vagal ganglia of Avil-Cre; Ai14 and Vglut2-Cre; Ai14 mice, tdTomato is expressed by PGP9.5-positive sensory neurons. (C) In the SCG of Avil-Cre; Ai14 mice, tdTomato is expressed in nearly all sympathetic ganglionic neurons positive for tyrosine hydroxylase (TH). In contrast, in the SCG of Vglut2-Cre; Ai14 mice, TH-positive ganglionic neurons lack tdTomato expression, although some tdTomato-positive fibers are observed within the SCG. Scale bars, 150 μm.

The Dst^Gt allele carries a multipurpose gene-trap cassette that enables both Cre-mediated inactivation and restoration of Dst through the inversion of the cassette. The mutant Dst^Gt-DO allele was inverted from functional Dst^Gt-inv allele by Cre recombinase (Figure 2A). For conditional gene-trap (cGT) experiments, male Avil-Cre; Dst^Gt-DO/WT mice were crossed with female Dst^{Gt-inv/Gt-inv} mice (Supplementary Figure 1). The Avil-Cre; Dst cGT mice (Avil-Cre; Dst^Gt-DO/Gt-inv) were obtained as one-fourth of the offspring, and mice of the other genotypes were used as littermate controls (Ctrl). To validate PNS neuron-selective inactivation of Dst expression, Dst transcripts were visualized by in situ hybridization (Figure 2B). In Ctrl mice, Dst mRNA was detected in sensory neurons of the DRG and vagal ganglia, as well as in sympathetic ganglionic neurons of the SCG. In contrast, Dst mRNA levels were markedly reduced in these neurons in Avil-Cre; Dst cGT mice.

Figure 2.

Histological analyses of sensory and autonomic ganglia in Avil-Cre; Ai14 mice. (A) A schematic representation of the Dst locus containing conditional gene-trap cassette. The gene-trap cassette consists of a splice acceptor (SA) sequence, the reporter gene βgeo, and polyadenylation (pA) termination signal. Pairs of inversely oriented target sites for Cre recombinase (loxP and lox5171: triangles) and FLP recombinase (Frt and F3: half circles) flank the gene-trap cassette. FLP- or Cre-mediated recombination irreversibly converts the mutant Dst^Gt allele into the functional Dst^Gt-inv allele, and the functional Dst^Gt-inv allele into the mutant Dst^Gt-DO allele. The Dst^Gt and Dst^Gt-DO alleles express nonfunctional Dst-β-geo, whereas the Dst^Gt-inv allele expresses functional Dst. (B) In situ hybridization analyses performed at 4 weeks of age. In Avil-Cre; Dst cGT mice, Dst mRNA levels are reduced in sensory neurons of the DRG and vagal ganglia, as well as in sympathetic ganglionic neurons of the SCG. (C,D) Neurodegenerative changes in the DRG, vagal ganglia, and SCG of Avil-Cre; Dst cGT mice at 4 weeks of age. Expression of the neuronal injury markers ATF3 (C) and Sprr1a (D) is increased in the DRG, vagal ganglia, and SCG of Avil-Cre; Dst cGT mice (arrowheads). Scale bars, 100 μm. (E,F) Quantitative analysis of the percentage of ATF3-positive (E) and Sprr1a-positive cells (F) in DRG, vagal ganglia, and SCG of Ctrl mice and Avil-Cre; Dst cGT mice (n = 4 Ctrl mice; n = 4 Avil-Cre; Dst cGT mice). * and *** denotes statistically significant difference at p < 0.05 and p < 0.005.

Sensory neurodegeneration is a pathological hallmark of dt mice (Duchen et al., 1964; Horie et al., 2016). We assessed neurodegenerative signs by analyzing the expression of ATF3 and Sprr1a in Avil-Cre; Dst cGT mice at 1 month of age (Figures 2C,D). ATF3 is a marker of neural injury due to its selective induction following nerve injury (Takeda et al., 2000; Tsujino et al., 2000; Kiryu-Seo et al., 2016). We previously reported that ATF3 expression is induced in DRG sensory neurons of Dst mutants (Yoshioka et al., 2024). Consistently, in Avil-Cre; Dst cGT mice, ATF3 expression was markedly induced in neurons of the DRG, vagal ganglia, and SCG, whereas it was scarcely detected in Ctrl mice (Figure 2C). Sprr1a is upregulated in DRG sensory neurons and spinal motor neurons following axotomy (Bonilla et al., 2002). We found that Sprr1a was also increased in sensory neurons of the DRG and vagal ganglia, as well as in sympathetic ganglionic neurons of the SCG in Avil-Cre; Dst cGT mice (Figure 2D). The expression of ATF3 and Sprr1a was quantified in DRG, vagal ganglia, and SCG of Ctrl and Avil-Cre; Dst cGT mice (Figures 2E,F). In all ganglia, the expression of both ATF3 and Sprr1a was significantly increased. However, the upregulation tended to be lower in the SCG compared with DRG and vagal ganglia. The expression of ATF3 and Sprr1a was also examined in parasympathetic ganglionic neurons of cardiac ganglia. These neural injury markers were scarcely induced in the systemic Dst-knockout (Dst GT) mice (Supplementary Figure 2) and Avil-Cre; Dst cGT mice (Supplementary Figure 3). Collectively, these histological analyses indicate that both PNS sensory (afferent) and sympathetic ganglionic (efferent) neurons of Avil-Cre; Dst cGT mice exhibit signs of neurodegeneration, while cardiac ganglionic neurons seem to be unaffected.

3.2. PNS neuron-selective inactivation of Dst gene causes dysregulated heart rate

Previously, we reported arrhythmias in isoform-specific Dst-b mutant mice, in which Dst expression is disrupted in cardiomyocytes (Yoshioka et al., 2022). These Dst-b mutants exhibited change in the ECG waveform, characterized by QT prolongation and premature contraction. In the present study, ECG recordings were obtained under isoflurane anesthesia in Avil-Cre; Dst cGT and Ctrl mice at 9–12 months of age (Supplementary Figure 4). ECG data were compared across three distinct epochs: induction, and the first and last minutes of a 5-min maintenance period. In the first minute of maintenance anesthesia, Avil-Cre; Dst cGT mice tended to exhibit highly variable heart rate rhythms compared with Ctrl mice (Figure 3A). HRV was quantified by the SD of RR intervals. The HRV of Avil-Cre; Dst cGT mice was significantly higher than that of Ctrl mice (genotype effect, F (1, 60) = 4.20, p < 0.05; epoch effect, F (2, 60) = 5.47, p < 0.01; genotype × epoch interaction, F (2, 60) = 4.12, p < 0.05, two-way ANOVA) (Figure 3B). The frequency of irregular pulse, defined as the number of instances exceeding ±3 × SD of RR intervals, was also significantly increased in Avil-Cre; Dst cGT mice compared with Ctrl mice (genotype effect, F (1, 60) = 12.04, p < 0.001; epoch effect, F (2, 60) = 8.18, p < 0.001; genotype × epoch interaction, F (2, 60) = 6.72, p < 0.01, two-way ANOVA) (Figure 3B). Although significant differences in HRV and irregular pulse were observed during immediate post-transition period, these effects diminished by the stabilized maintenance period. Two-way ANOVA revealed that Avil-Cre; Dst cGT mice exhibit a significantly widened QRS complex (genotype effect, F (1, 60) = 14.63, p < 0.001; epoch effect, F (2, 60) = 0.07, p > 0.05; genotype × epoch interaction, F (2, 60) = 0.48, p > 0.05, two-way ANOVA) and a shortened PR interval (genotype effect, F (1, 60) = 16.38, p < 0.001; epoch effect, F (2, 60) = 0.00, p > 0.05; genotype × epoch interaction, F (2, 60) = 0.68, p > 0.05, two-way ANOVA) compared with Ctrl mice. However, no significant main effect of epoch or genotype × epoch interaction for these parameters was observed. In contrast, no significant differences were observed in RR and QT intervals with respect to either genotype or epoch, and no interaction was detected (Figure 3B). Collectively, these findings indicate that loss of Dst in PNS sensory and sympathetic ganglionic neurons results in dysregulated heart rhythms and significant conduction alterations.

Figure 3.

Electrocardiogram recordings from Avil-Cre; Dst cGT mice under anesthesia. (A) Representative electrocardiogram (ECG) traces from Ctrl and Avil-Cre; Dst cGT mice under anesthesia. Arrows indicate abnormal skipping of P waves. (B) Quantification of mean RR intervals, HRV, frequency of irregular pulse, PR interval, QRS duration, and QT interval (n = 12 Ctrl mice; n = 10 Avil-Cre; Dst cGT mice, at 9–12 months of age). ECG was quantified in three epochs: (i) 1-min of the induction anesthesia period, (ii) first 1-min of maintenance anesthesia period, and (iii) last 1-min of the 5-min maintenance period. *, **, and *** denotes statistically significant difference at p < 0.05, p < 0.01, and p < 0.001 and ns means not statistically significant (p > 0.05), using two-way ANOVA. Data are presented as mean ± SD.

Next, we performed pathological analyses of the heart tissues in Avil-Cre; Dst cGT mice. We previously reported that loss of the Dst-b isoform from cardiomyocytes leads to cardiomyopathy accompanied by upregulation of the heart failure marker gene natriuretic peptide A (Nppa) (Yoshioka et al., 2022). Here, we confirmed upregulation of the Nppa transcript in the myocardium of left ventricle from Dst-b mutants (Dst-b^{E2610Ter/E2610Ter}) at 2 years old (Figure 4A). In contrast, the Nppa transcript was sporadically detected in the myocardium of age-matched Avil-Cre; Dst cGT mice. Cardiac fibrosis was assessed by Masson’s trichrome staining (Figure 4B). A marked increase in the connective tissue was observed in Dst-b^{E2610Ter/E2610Ter} mice. In contrast, collagen deposition was focal and occurred to a lesser extent in Avil-Cre; Dst cGT mice. We have also reported that protein aggregate formation is a pathological hallmark of cardiomyopathy in Dst-b^{E2610Ter/E2610Ter} mice (Yoshioka et al., 2022). Previous RNA sequencing analyses revealed increased expression of chaperone protein-coding genes involved in unfolded protein response (UPR). Here, we performed immunohistochemical analyses of chaperone proteins HspB1/Hsp25 and HspA1L, which are upregulated at the RNA level in Dst-b^{E2610Ter/E2610Ter} mice (Yoshioka et al., 2022). HspB1/Hsp25 and HspA1L densely aggregated in Dst-b^{E2610Ter/E2610Ter} cardiomyocytes, displaying distinct staining patterns. HspB1/Hsp25 was diffusely distributed throughout the cytoplasm (Figure 4C), whereas HspA1L-positive aggregates appeared as small punctate signals (Figure 4D). p62 was also observed as dot-like aggregates within the nuclei of Dst-b^{E2610Ter/E2610Ter} cardiomyocytes (Yoshioka et al., 2022), and immunofluorescent staining confirmed nuclear localization of HspA1L-positive aggregates (Supplementary Figure 5). These findings suggest that alterations in chaperone protein function may contribute to protein aggregate formation in Dst-b^{E2610Ter/E2610Ter} mice. In the heart tissues of Avil-Cre; Dst cGT mice, aggregations of HspB1/Hsp25 and HspA1L were scarcely observed. These data suggest that the loss of the Dst-b isoform leads to cardiomyocyte dysfunction and degeneration, whereas the conditional loss of the Dst-a isoform in the PNS circuit does not directly link to severe damage to cardiomyocytes themselves. Therefore, the dysregulation of heart rate in Avil-Cre; Dst cGT mice is likely caused by neural circuit abnormalities rather than by cardiomyopathy.

Figure 4.

Pathological analyses of cardiac tissues. (A) Nppa in situ hybridization in the heart at 2 years of age. Nppa mRNA expression is upregulated in the left ventricular (LV) myocardium of Dst-b^{E2610Ter/E2610Ter} mice, but not remarkable in Avil-Cre; Dst cGT mice or Ctrl mice. Dotted lines indicate tissue boundaries. (B) Masson’s trichrome staining showed a sever fibrosis in the myocardium of Dst-b^{E2610Ter/E2610Ter} mice (yellow arrows). Focal collagen depositions were observed in the myocardium of Avil-Cre; Dst cGT mice. (C,D) Immunohistochemical analyses of heart sections from Ctrl, Avil-Cre; Dst cGT, and Dst-b^{E2610Ter/E2610Ter} mice using antibodies against HSPB1/HSP25 (C) and HSPA1L (D). HSPB1/HSP25 accumulates in the cytoplasm of cardiomyocytes of Dst-b^{E2610Ter/E2610Ter} mice (arrows). HSPA1L accumulates in cardiomyocytes as punctate signals (arrowheads). Scale bars, 1,000 μm (A), 100 μm (B), and 50 μm (C,D).

3.3. Disruption of PNS afferent circuit leads to arrhythmias

Our data suggest that disruption of the PNS neural circuit underlies arrhythmias in Dst mutant mice. However, it remains unclear whether abnormalities in the afferent or efferent circuits are involved in the dysregulation of heart rate. To address this issue, we next used Vglut2-Cre mice to selectively inactivate the Dst expression in sensory neurons, because Vglut2-Cre-mediated recombination occurs in sensory neurons but not in sympathetic ganglionic neurons of the PNS (Niu et al., 2020). Although Avil has been accepted to be specifically expressed by PNS neurons (Hunter et al., 2018), vesicular glutamate transporter 2 (Vglut2) is widely expressed by glutamatergic neurons in the central nervous system (CNS) (Kaneko and Fujiyama, 2002). Therefore, we exclusively compare the regions of Cre-mediated recombination in the whole brain and spinal cord of Avil-Cre; Ai14 and Vglut2-Cre; Ai14 after tissue clearing using CUBIC (Supplementary Figure 6). In the brain and spinal cord of Avil-Cre; Ai14, tdTomato signals were localized in axonal trajectories derived from sensory neurons of the DRG and trigeminal ganglia. In contrast, in Vglut2-Cre; Ai14, tdTomato-positive cells were distributed throughout the gray matter of the spinal cord, and tdTomato signals were observed throughout the cerebral cortex, where PNS sensory neurons do not directly project. These observations confirmed that Avil-Cre is specific to PNS neurons, while Vglut2-Cre is active in both CNS and PNS neurons. In the PNS of Vglut2-Cre; Ai14 mice, tdTomato was expressed in sensory neurons of the DRG (Figure 1A) and vagal ganglia (Figure 1B), but not in sympathetic ganglionic neurons of the SCG (Figure 1C). In the SCG, tdTomato was detected only in nerve fibers. These data indicate that Cre-mediated recombination occurs in PNS afferent circuits but not in PNS efferent circuits of Vglut2-Cre mice, as expected. Qualitative histological analyses revealed that neural injury markers ATF3 and Sprr1a were induced in sensory neurons of the DRG and vagal ganglia in Vglut2-Cre; Dst cGT mice, while these markers were scarcely expressed in sympathetic ganglionic neurons of the SCG (Figure 5). These findings indicate that PNS afferent circuits are selectively disrupted in Vglut2-Cre; Dst cGT mice, whereas PNS efferent circuits remain unaffected.

Figure 5.

Inactivation of Dst expression in sensory neurons causes sensory neurodegeneration. Histological analysis of neurodegenerative changes in the DRG, vagal ganglia, and SCG of Vglut2-Cre; Dst cGT mice at 4 weeks of age. Expression of the neuronal injury markers ATF3 and Sprr1a is increased in the DRG and vagal ganglia (arrowheads). In contrast, ATF3 and Sprr1a are scarcely expressed in the SCG of Vglut2-Cre; Dst cGT mice. Scale bars, 100 μm.

Finally, we recorded ECG from Vglut2-Cre; Dst cGT and Ctrl mice at 11–13 months of age. Similar to Avil-Cre; Dst cGT mice, some Vglut2-Cre; Dst cGT mice exhibited elevated HRV immediately after the transition to maintenance anesthesia (Figure 6A). Statistically, Vglut2-Cre; Dst cGT mice exhibited mild but significant increases in HRV (genotype effect, F (1, 51) = 7.47, p < 0.01; epoch effect, F (2, 51) = 4.87, p < 0.05; genotype × epoch interaction, F (2, 51) = 0.58, p > 0.05, two-way ANOVA) and the frequency of irregular pulse (genotype effect, F (1, 51) = 10.05, p < 0.01; epoch effect, F (2, 51) = 1.69, p > 0.05; genotype × epoch interaction, F (2, 51) = 1.69, p > 0.05, two-way ANOVA), as well as a significant shortening of the PR interval compared with Ctrl mice (genotype effect, F (1, 51) = 5.63, p < 0.05; epoch effect, F (2, 51) = 3.05, p > 0.05; genotype × epoch interaction, F (2, 51) = 2.04, p > 0.05, two-way ANOVA) (Figure 6B). QRS complex of Vglut2-Cre; Dst cGT mice remained unchanged (Figure 6B). Furthermore, unlike the Avil-Cre line, Vglut2-Cre; Dst cGT mice displayed significant alterations in RR (genotype effect, F (1, 51) = 13.78, p < 0.001; epoch effect, F (2, 51) = 3.78, p < 0.05; genotype × epoch interaction, F (2, 51) = 0.37, p > 0.05, two-way ANOVA) and QT intervals (genotype effect, F (1, 51) = 4.57, p < 0.05; epoch effect, F (2, 51) = 0.07, p > 0.05; genotype × epoch interaction, F (2, 51) = 0.45, p > 0.05, two-way ANOVA) (Figure 6B). These data suggest that Vglut2-Cre-mediated Dst deficiency also leads to rhythmic instability and conduction changes. The specific impact on ventricular depolarization and repolarization differs from that observed in Avil-Cre; Dst cGT mice. Since isoflurane is known to affect the autonomic nervous system (Kato et al., 1992), we investigated whether cardiac rhythm instability occurs in the conscious state (Supplementary Figure 7). We found that one out of the three Vglut2-Cre; Dst cGT mice exhibited rhythmic irregularities in the conscious state. These results support the idea that disruption of the PNS afferent circuit, rather than degeneration of sympathetic ganglionic neurons, is a major contributor to dysregulation of the heart rhythm.

Figure 6.

Electrocardiogram recordings from Vglut2-Cre; Dst cGT mice under anesthesia. (A) Representative ECG traces from Ctrl and Vglut2-Cre; Dst cGT mice under anesthesia. Arrows indicate abnormal skipping of P waves. (B) Quantification of mean RR intervals, HRV, frequency of irregular pulse, PR interval, QRS duration, and QT interval (n = 10 Ctrl mice; n = 9 Vglut2-Cre; Dst cGT mice, at 11–13 months of age). ECG was quantified in three epochs: (i) 1 min of the induction anesthesia period, (ii) First 1-min of maintenance anesthesia period, and (iii) The final 1 min of the 5-min maintenance period. *, **, and *** denotes statistically significant difference at p < 0.05, p < 0.01, and p < 0.001 and ns means not statistically significant (p > 0.05), using two-way ANOVA. Data are presented as mean ± SD.

4. Discussion

The Significance of Disruption of PNS Afferent and Efferent Circuits in Cardiac Dysregulation.

The HSANs comprise complex group of inherited disorders primarily affecting sensory and autonomic nervous systems. Patients with HSANs exhibit multimodal somatosensory impairments, including defects in touch, pain, and proprioception. While somatosensory dysfunction has been extensively investigated, the pathophysiology of visceral dysfunction remains relatively unexplored. Dysregulation of the autonomic nervous system is generally accepted to impair visceral function, leading to a wide range of clinical manifestations such as arrhythmias, orthostatic hypotension, and gastrointestinal motility disorders (Sakae et al., 2001; Thireau et al., 2017). Recent studies using animal models have further elucidated the intricate relationship between autonomic dysfunction and cardiac abnormalities. For example, Meis1-deficient mice exhibit abnormal development of sympathetic ganglionic neurons and increased HRV, implicating sympathetic dysfunction in cardiac irregularities (Bouilloux et al., 2016).

The complex mechanisms underlying visceral dysfunction have been investigated using the Elp1 mutant mouse, a model of familial dysautonomia (FD; HSAN-III). Conditional deletion of the Elp1 gene in sensory and sympathetic ganglionic neurons results in impaired target tissue innervation, supporting the notion that abnormal development of sensory and autonomic neural circuits is a fundamental mechanism of visceral dysfunction (Jackson et al., 2014). The gastrointestinal system is regulated by both extrinsic innervations from the vagus nerve, sympathetic ganglia, and dorsal root ganglia, as well as by the intrinsic enteric nervous system (Sharkey and Mawe, 2023). Conditional deletion of Elp1 from PNS neurons disrupts normal formation of the enteric nervous system and target innervation of the intestinal mucosa and smooth muscle (Chaverra et al., 2024). Such abnormalities perturb the intestinal epithelial barrier and may underlie gastrointestinal motility disorders in FD patients. Collectively, these animal studies highlight the diverse and complex neural circuit mechanisms contributing to visceral dysfunction in HSANs.

As a model for HSAN-VI, Dst mutant mice have also been used to explore the mechanisms underlying gastrointestinal dysfunction (Lynch-Godrei et al., 2020). Dst mutants exhibit slowed gastrointestinal motility and thinning of the colonic mucus layer, despite apparently preserved enteric nervous system architecture. Instead, Dst mutants show evidence of neurodegeneration in the vagus nerve and vagal ganglia, with little to no degeneration in sympathetic ganglia (Lynch-Godrei et al., 2020), suggesting that impaired parasympathetic control contributes to gastrointestinal dysfunction in HSAN-VI. However, the role of visceral afferent circuit abnormalities has remained unclear. In the present study, we demonstrated that PNS neuron-selective ablation of Dst gene (Avil-Cre; Dst cGT) induces upregulations of neural injury markers ATF3 and Sprr1a in sensory neurons of the DRG and vagal ganglia, as well as sympathetic ganglionic neurons of the SCG. Coinciding with previous findings (Lynch-Godrei et al., 2020), neurodegenerative signs were more pronounced in sensory neurons of the vagal ganglia than in sympathetic ganglionic neurons of the SCG. Avil-Cre; Dst cGT mice exhibited arrhythmias characterized by increased HRV. Notably, milder but similar higher HRV and irregular pulses were observed in Vglut2-Cre; Dst cGT mice, in which degeneration was restricted to PNS sensory neurons, while sympathetic ganglionic neurons remained intact. It suggests that the increased HRV is primarily driven by the degeneration of PNS sensory neurons. In contrast, the differences in ventricular depolarization and repolarization between the two models may result from distinct recombination in other regions, including the CNS, rather than sensory circuits alone. Furthermore, while Vglut2-Cre does not induce direct recombination within the autonomic neurons (Chang et al., 2015), it may indirectly modulate autonomic nervous system since Vglut2-positive interneurons are known to regulate structural and functional plasticity of sympathetic nervous systems after spinal cord injury (Noble et al., 2022). These conditional gene-targeting strategies support the concept that degeneration of PNS sensory neurons leads to the disturbance of visceral afferent circuits and subsequent dysregulation of heart rhythms via disruption of interactions between visceral afferent and efferent circuits. However, it should be noted that the relationship observed between these specific regions of neurodegeneration and the onset of arrhythmia remains correlative, and there are technical limitations in establishing a definitive causative link within the current framework. In recent years, the use of viral vectors and optogenetics has allowed precise mapping of the peripheral neural circuits involved in heart rate control (Rajendran et al., 2019). In future studies, more localized and temporal manipulation of Dst expression will elucidate the direct causal relationship between sensory circuit disruption and arrhythmogenesis. Furthermore, it is well-established that isoflurane anesthesia disrupts autonomic nervous system balance (Kato et al., 1992). While the occurrence of arrhythmias in the awake state indicates that autonomic dysfunction and rhythm disturbances do not always manifest concurrently, the significantly increased frequency of these events under isoflurane anesthesia suggests that Dst mutant mice possess a heightened vulnerability to autonomic stressors. This increased susceptibility likely reflects impaired homeostatic resilience, highlighting the critical role of sensory circuits in maintaining visceral homeostasis.

4.1. PNS afferent control of heart rate via multiple feedback circuits

Visceral sensation is generally attributed to sensory neurons in vagal ganglia (Waise et al., 2018; Ichiki et al., 2022; Prescott and Liberles, 2022). Recent optogenetic and viral tracing studies have demonstrated that afferent signals conveyed via the vagus nerve enhance parasympathetic tone and suppress sympathetic activity to the heart through the CNS pathways (Rajendran et al., 2019). In contrast, other studies have identified tropomyosin receptor kinase C (TrkC)-positive sensory neurons in the DRG that regulate blood pressure and heart rate via sympathetic nervous system pathways (Morelli et al., 2021). Together, these findings indicate that PNS sensory neurons control cardiovascular function through multiple feedback circuits involving the autonomic nervous system. In the present study, sensory neuron-selective loss of the Dst leads to severe degeneration of sensory neurons in both the vagal ganglia and DRG, suggesting a simultaneous disruption of the visceral feedback circuit through these distinct routes. DRG neurons are derived exclusively from the neural crest, whereas vagal ganglia arise from a combination of neural crest cells and ectodermal placodes. This dual developmental origin may contribute to the pathological heterogeneity observed in visceral sensory deficits among HSANs. Elucidating how these developmental differences influence the differential vulnerability of sensory neuron populations will be an important direction for future research.

4.2. The mechanisms of pathogenesis of visceral dysfunctions in HSAN

The pathophysiology of visceral dysfunction in HSANs, including cardiovascular reflex abnormalities, gastrointestinal dysfunction, and respiratory failure, is multifactorial and involves complex interactions among multiple organs. The Dst gene encodes tissue-selective isoforms DST-a, DST-b, and DST-e expressed predominantly in neural, muscular, and cutaneous tissues, respectively (Künzli et al., 2016). These Dst isoforms play critical roles in maintaining tissue homeostasis (Yoshioka, 2024). Our initial analysis of Dst-b mutant mice predicted the presence of muscle pathology (Yoshioka et al., 2022), leading to the identification of patients with congenital myopathy and cardiomyopathy associated with DST-b mutations (Jacob et al., 2025). The Dst-b mutation induces protein aggregate formation accompanied by accumulation of chaperone proteins associated with the UPR. Consequently, Dst-b mutant mice exhibit arrhythmias, including QT prolongation and premature contraction with protein aggregate cardiomyopathy (Yoshioka et al., 2022). In contrast, PNS neuron-selective Dst cGT mice (Avil-Cre; Dst cGT and Vglut2-Cre; Dst cGT) displayed sinus arrhythmias without the formation of protein aggregates. Although Avil-Cre; Dst cGT mice exhibited less severe cardiomyocyte damage compared to Dst-b mutant mice, the presence of mild fibrosis suggests that the PNS may contribute to the maintenance of cardiomyocytes. Therefore, the mechanisms by which peripheral nerve dysfunction mediates myocardial damage remain an important topic for future research. These findings suggest that DST mutations can cause arrhythmia through multiple mechanisms, including intrinsic cardiomyocyte dysfunction and the disruption of neural circuits regulating cardiac functions. Respiratory failure has also been also reported as a cause of juvenile death in patients with severe form of HSAN-VI (Edvardson et al., 2012; Ahmad et al., 2025). Notably, vagal ganglia contain sensory neurons that project to both the cardiovascular and respiratory systems, potentially mediating coordination between respiration and heart rate (Devarajan et al., 2022). Given the established association between cardiac arrhythmias and respiratory control, this interaction should be considered in the pathophysiological mechanisms underlying arrhythmias in HSANs. Combining these findings, it is necessary to elucidate visceral dysfunction in HSANs from a comprehensive perspective that integrates the roles of causative genes, neural circuits, and multi-organ interactions.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This project was funded by a grant from JSPS (20 K15912, and 23 K06317 to NY, 21 K10169, and 25 K13088 to MK, 15H04667, 18H02592, and 21H02652 to HT), Grant-in-Aid for Scientific Research on Innovative Areas, “Non-linear Neuro-oscillology” (18H04939 to HT), The Nakatani Foundation (NY), Nippon Shinyaku Research Grant (NY), SENSHIN Medical Research Foundation (NY), Nagai Promotion Foundation for Science of Perception (NY and HT), UNION TOOL Scholarship Foundation (NY), Takeda Science Foundation (NY), Mochida Memorial Foundation for Medical and Pharmaceutical Research (NY), Kato Memorial Bioscience Foundation (NY), Niigata University Interdisciplinary Research Grant (NY), the Uehara Memorial Foundation (HT), and a grant for Interdisciplinary Joint Research Project from Brain Research Institute from Niigata University (HT).

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The animal studies were approved by Internal Review Board of Niigata University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

Author contributions

NY: Conceptualization, Funding acquisition, Investigation, Project administration, Visualization, Writing – original draft, Data curation, Writing – review & editing. MK: Investigation, Conceptualization, Data curation, Funding acquisition, Methodology, Visualization, Writing – original draft, Writing – review & editing. KT: Investigation, Methodology, Writing – review & editing. TI: Investigation, Writing – review & editing. YT: Resources, Writing – review & editing. HF: Resources, Writing – review & editing. MU: Resources, Writing – review & editing. HO: Supervision, Writing – review & editing. IK: Supervision, Writing – review & editing. HT: Supervision, Writing – review & editing, Conceptualization, Funding acquisition, Methodology, Resources.

Conflict of interest

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The authors HF and MU declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncir.2026.1777115/full#supplementary-material