Human induced pluripotent stem cell-derived cardiac myocytes and sympathetic neurons in disease modelling.

Human induced pluripotent stem cells (hiPSC) offer an unprecedented opportunity to generate model systems that facilitate a mechanistic understanding of human disease. Current differentiation protocols are capable of generating cardiac myocytes (hiPSC-CM) and sympathetic neurons (hiPSC-SN). However, the ability of hiPSC-derived neurocardiac co-culture systems to replicate the human phenotype in disease modelling is still in its infancy. Here, we adapted current methods for efficient and replicable induction of hiPSC-CM and hiPSC-SN. Expression of cell-type-specific proteins were confirmed by flow cytometry and immunofluorescence staining. The utility of healthy hiPSC-CM was tested with pressor agents to develop a model of cardiac hypertrophy. Treatment with angiotensin II (AngII) resulted in: (i) cell and nuclear enlargement, (ii) enhanced fetal gene expression, and (iii) FRET-activated cAMP responses to adrenergic stimulation. AngII or KCl increased intracellular calcium transients in hiPSC-SN. Immunostaining in neurocardiac co-cultures demonstrated anatomical innervation to myocytes, where myocyte cytosolic cAMP responses were enhanced by forskolin compared with monocultures. In conclusion, human iPSC-derived cardiac myocytes and sympathetic neurons replicated many features of the anatomy and (patho)physiology of these cells, where co-culture preparations behaved in a manner that mimicked key physiological responses seen in other mammalian systems. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Abnormal Cyclic Nucleotide Signaling at the Outer Mitochondrial Membrane In Sympathetic Neurons During the Early Stages of Hypertension.

BACKGROUND: Disruption of cyclic nucleotide signaling in sympathetic postganglionic neurons contributes to impaired intracellular calcium handling (Ca2+) and the development of dysautonomia during the early stages of hypertension, although how this occurs is poorly understood. Emerging evidence supports the uncoupling of signalosomes in distinct cellular compartments involving cyclic nucleotide-sensitive PDEs (phosphodiesterases), which may underpin the autonomic phenotype in stellate neurons. METHODS: Using a combination of single-cell RNA sequencing together with Forster resonance energy transfer-based sensors to monitor cyclic adenosine 3',5'-monophosphate, PKA (protein kinase A)-dependent phosphorylation and cGMP (cyclic guanosine 3',5'-monophosphate), we tested the hypothesis that dysregulation occurs in a sub-family of PDEs in the cytosol and outer mitochondrial membrane of neurons from the stellate ganglion. RESULTS: PDE2A, 6D, 7A, 9A genes were highly expressed in young Wistar neurons and also conserved in neurons from spontaneously hypertensive rats (SHRs). In stellate neurons from prehypertensive SHRs, we found the levels of cyclic adenosine 3',5'-monophosphate and cGMP at the outer mitochondrial membrane were decreased compared with normal neurons. The reduced cyclic adenosine 3',5'-monophosphate response was due to the hydrolytic activity of overexpressed PDE2A2 located at the mitochondria. Normal cyclic adenosine 3',5'-monophosphate levels were re-established by inhibition of PDE2A. There was also a greater PKA-dependent phosphorylation in the cytosol and at the outer mitochondrial membrane in spontaneously hypertensive rat neurons, where this response was regulated by protein phosphatases. The cGMP response was only restored by inhibition of PDE6. CONCLUSIONS: When taken together, these results suggest that site-specific inhibition of PDE2A and PDE6D at the outer mitochondrial membrane may provide a therapeutic target to ameliorate cardiac sympathetic impairment during the onset of hypertension.

Macrophages Can Drive Sympathetic Excitability in the Early Stages of Hypertension.

Hypertension is a major health burden worldwide with many cases resistant to current treatments. Hyperactivity of the sympathetic nervous contributes to the etiology and progression of the disease, where emerging evidence suggests that inflammation may underpin the development of sympathetic dysautonomia. This study examined whether macrophages could drive the sympathetic phenotype in Spontaneously Hypertensive Rats (SHR) before animals develop high pressure. Stellate neurons from wild-type control Wistar rats and SHRs were co-cultured with blood leukocytes from their own strain, and also crossed cultured between strains. The calcium transient response to nicotinic stimulation was recorded using Fura-2 calcium imaging, where SHR neurons had a greater calcium transient compared with Wistar neurons. However, when co-cultured with leukocytes, Wistar neurons began to phenocopy the SHR sympathetic hyperactivity, while the SHR neurons themselves were unaltered. Resident leukocyte populations of the SHR and Wistar stellate ganglia were then compared using flow cytometry, where there was a shift in monocyte-macrophage subset proportions. While classical monocyte-macrophages were predominant in the Wistar, there were relatively more of the non-classical subset in the SHR, which have been implicated in pro-inflammatory roles in a number of diseases. When bone marrow-derived macrophages (BMDMs) were co-cultured with stellate neurons, they made Wistar neurons recapitulate the SHR nicotinic stimulated calcium transient. Wistar BMDMs however, had no effect on SHR neurons, even though SHR BMDMs increased SHR neuron responsiveness further above their hyper-responsive state. Taken together, these findings show that macrophages can be potent enhancers of sympathetic neuronal calcium responsiveness, and thus could conceivably play a role in peripheral sympathetic hyperactivity observed in the early stages of hypertension.

Angiotensin peptide synthesis and cyclic nucleotide modulation in sympathetic stellate ganglia.

Chronically elevated angiotensin II is a widely-established contributor to hypertension and heart failure via its action on the kidneys and vasculature. It also augments the activity of peripheral sympathetic nerves through activation of presynaptic angiotensin II receptors, thus contributing to sympathetic over-activity. Although some cells can synthesise angiotensin II locally, it is not known if this machinery is present in neurons closely coupled to the heart. Using a combination of RNA sequencing and quantitative real-time polymerase chain reaction, we demonstrate evidence for a renin-angiotensin synthesis pathway within human and rat sympathetic stellate ganglia, where significant alterations were observed in the spontaneously hypertensive rat stellate ganglia compared with Wistar stellates. We also used Förster Resonance Energy Transfer to demonstrate that administration of angiotensin II and angiotensin 1-7 peptides significantly elevate cyclic guanosine monophosphate in the rat stellate ganglia. Whether the release of angiotensin peptides from the sympathetic stellate ganglia alters neurotransmission and/or exacerbates cardiac dysfunction in states associated with sympathetic over activity remains to be established.

Obesity: a neuroimmunometabolic perspective.

Neuroimmunology and immunometabolism are burgeoning topics of study, but the intersection of these two fields is scarcely considered. This interplay is particularly prevalent within adipose tissue, where immune cells and the sympathetic nervous system (SNS) have an important role in metabolic homeostasis and pathology, namely in obesity. In the present Review, we first outline the established reciprocal adipose-SNS relationship comprising the neuroendocrine loop facilitated primarily by adipose tissue-derived leptin and SNS-derived noradrenaline. Next, we review the extensive crosstalk between adipocytes and resident innate immune cells as well as the changes that occur in these secretory and signalling pathways in obesity. Finally, we discuss the effect of SNS adrenergic signalling in immune cells and conclude with exciting new research demonstrating an immutable role for SNS-resident macrophages in modulating SNS-adipose crosstalk. We posit that the latter point constitutes the existence of a new field - neuroimmunometabolism.

FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons.

Several FRET (fluorescence resonance energy transfer)-based biosensors for intracellular detection of cyclic nucleotides have been designed in the past decade. However, few such biosensors are available for cGMP, and even fewer that detect low nanomolar cGMP concentrations. Our aim was to develop a FRET-based cGMP biosensor with high affinity for cGMP as a tool for intracellular signaling studies. We used the carboxyl-terminal cyclic nucleotide binding domain of Plasmodium falciparum cGMP-dependent protein kinase (PKG) flanked by different FRET pairs to generate two cGMP biosensors (Yellow PfPKG and Red PfPKG). Here, we report that these cGMP biosensors display high affinity for cGMP (EC50 of 23 ± 3 nM) and detect cGMP produced through soluble guanylyl cyclase and guanylyl cyclase A in stellate ganglion neurons and guanylyl cyclase B in cardiomyocytes. These biosensors are therefore optimal tools for real-time measurements of low concentrations of cGMP in living cells.

Phosphodiesterase 2A as a therapeutic target to restore cardiac neurotransmission during sympathetic hyperactivity.

Elevated levels of brain natriuretic peptide (BNP) are regarded as an early compensatory response to cardiac myocyte hypertrophy, although exogenously administered BNP shows poor clinical efficacy in heart failure and hypertension. We tested whether phosphodiesterase 2A (PDE2A), which regulates the action of BNP-activated cyclic guanosine monophosphate (cGMP), was directly involved in modulating Ca2+ handling from stellate ganglia (SG) neurons and cardiac norepinephrine (NE) release in rats and humans with an enhanced sympathetic phenotype. SG were also isolated from patients with sympathetic hyperactivity and healthy donor patients. PDE2A activity of the SG was greater in both spontaneously hypertensive rats (SHRs) and patients compared with their respective controls, whereas PDE2A mRNA was only high in SHR SG. BNP significantly reduced the magnitude of the calcium transients and ICaN in normal Wistar Kyoto (WKY) SG neurons, but not in the SHRs. cGMP levels stimulated by BNP were also attenuated in SHR SG neurons. Overexpression of PDE2A in WKY neurons recapitulated the calcium phenotype seen in SHR neurons. Functionally, BNP significantly reduced [3H]-NE release in the WKY rats, but not in the SHRs. Blockade of overexpressed PDE2A with Bay 60-7550 or overexpression of catalytically inactive PDE2A reestablished the modulatory action of BNP in SHR SG neurons. This suggests that PDE2A may be a key target in modulating the action of BNP to reduce sympathetic hyperactivity.