Rationale: The sympathetic branch of the autonomic nervous system (ANS) operates continuous control on the function and structure of cardiac cells (Bers et al., 2009). In basal conditions, neuronal input to sino-atrial node (SAN) cardiomyocytes (CM) is responsible for the fine regulation of heart rate (HR), and in parallel, it influences the balance between protein synthesis and degradation in working CMs, thus determining the resting cellular trophism (Zaglia et al., 2013). It is also commonly appreciated that sympathetic neurons (SNs) are rapidly activated upon strenuous exercise or emotional stresses characterizing the so-called ‘fight-or-flight’ response, resulting in the recruitment of maximal cardiac performance through positive inotropic and chronotropic effects (Li et al., 2000). While these general mechanisms of regulation of heart physiology are commonly recognized and have been thoroughly investigated in the last decades in both normal and disease conditions (Franzoso et al., 2016), the relationship between the fine anatomy of the myocardial neuronal network and its function, as well as the biophysics of the neuro-effector communication remain largely uncovered.
Purposes: In our initial studies, we used various imaging methods to investigate the morphological aspects of myocardial innervation. Our results demonstrate that the heart of most mammals, including humans, is highly innervated by SN processes, which distribute throughout the different heart regions with a conserved, specie-specific pattern, and display regular varicosities (i.e. active neurotransmitter release sites), which appear in close contact with the target CM membranes. Furthermore, ultrastructural analysis showed that such contacts have features similar to those described for the well-known neuromuscular junction (NMJ), which, remarkably, include the accumulation of mitochondria in the “presynaptic” varicosities (Slater, 2003; Levitan et al., 2015).
This data prompted us to study:
i) the biophysics of neuro-cardiac communication, which was addressed using both in vitro and in vivo models, with the aim to determine the role of direct intercellular contact in the dynamics of neuro-effector coupling;
ii) whether dysfunction in SN mitochondria, as addressed in a newly developed murine model of Optic Atrophy Factor-1 (Opa1) haploinsufficiency (TOH-Opa1+/-), may affect the neurogenic control of the heart.
Results: i) Dynamics of neuro-effector coupling at ‘cardiac sympathetic’ synapses.
The aim of this study has been to investigated the dynamics of SN/cardiomyocyte intercellular signaling communication, both by FRET-based imaging of cAMP in co-cultures, as a readout of cardiac β-AR activation, and in vivo, using optogenetics in transgenic mice with SN-specific expression of Channelrhodopsin-2. We demonstrate that SNs and cardiomyocytes interact at specific sites both in the human and rodent heart, and in co-cultures. Accordingly, neuronal activation elicited intracellular cAMP increases only in directly contacted myocytes and cell-cell coupling utilized a junctional extracellular signaling domain with elevated noradrenaline concentration. In the living mouse, optogenetic activation of cardiac SNs, innervating the sino-atrial node, resulted in the instantaneous chronotropic effect, which shortened the heartbeat interval with single beat precision. The dose of the β-blocker propranolol inhibiting the effect of photoactivation was much higher than that blocking circulating catecholamines, thus indicating that sympathetic neurotransmission in the heart occurs at locally elevated noradrenaline concentration. Our in vitro and in vivo data suggest that the control of cardiac function by SNs, thanks to the establishment of a specific intercellular junctional-site, relies on ‘quasi-synaptic’ intercellular communication. The closely juxtaposed membranes of neurons and cardiomyocytes outline an extracellular signaling domain allowing activation of the β-ARs localized within the junctional space high with [NE]. The very small volume of such domain allows a single neuronal action potential to release a [NE] sufficient to trigger detectable cAMP increase in the coupled cardiomyocytes.
ii) Role of the mitochondrial protein Opa1 in the regulation of the cardiac SN physiology.
In the study of neuro-cardiac interactions described above, we were intrigued by the observation that mitochondria accumulated in SN varicosities, and specifically concentrated in the subspace of the presynaptic membrane. Neuronal mitochondria are fundamental for several cellular functions, including neuro-exocytosis, neurotransmitter reuptake and maintenance of neuronal process trophism, but their specific role in cardiac SNs is largely unexplored.
We thus sought to determine whether dysfunctional mitochondria would compromise the neurogenic control of the heart. To this aim, we exploited a murine model generated in our laboratory, characterized by the haploinsufficiency of Opa1 gene (Hoppins et al., 2007), selectively in SNs. Opa1 is a key protein implicated in mitochondrial dynamics, and its deficiency causes an inherited neurodegenerative disease characterized by retinal ganglion cell death known as Autosomal Dominant Optic Atrophy (ADOA), leading to visual loss. Interestingly, ADOA patients also display peripheral neuropathy and cardiac rhythm abnormalities (Spiegel et al., 2016) suggesting the hypothesis that dysfunctional mitochondria may affect not only central but also peripheral neurons, and remarkably, the autonomic neurons innervating the heart (Yu-Wai Man et al., 2016). To address this hypothesis, we focused on cardiac sympathetic innervation in both adult and aged Opa1 haploinsufficient mice (TOH-Opa1+/-) mice, which was studied using morphological and functional assays.
Our data demonstrated that Opa1 haploinsufficiency leads to a decrease in cSN density, which starts in the adulthood but it is also present during ageing. This is accompanied to alterations in cSN distribution patterning and morphology. Cardiac dysinnervation in TOH-Opa1+/- mice results in a significant decrease in heart rate variability and increased propensity to arrhythmias developments. Consistently, we detected decreased SN density in skin biopsies from ADOA patients, which progresses during ageing.
Thus we can conclude that the Opa1 is essential for cSN homeostasis and indicate that its haploinsufficiency leads to cSN degeneration. The cSN dysinnervation causes the dysfunction of the extrinsic control of cardiac rhythm. The mechanisms responsible for Opa1 haploinsufficiency-dependent cSN degeneration will be assessed in vitro, with a focus on the NGF signalling. To translate our findings to the human pathology, we will analyse SN phenotype in skin biopsies from ADOA patients.
Conclusions: Collectively, the data from these two projects, pose the bases for future studies aimed at defining whether a primary alteration in the SN-CM contact contribute to the pathogenesis of several cardiovascular disorders and at clarifying the molecular mechanisms whereby defective mitochondrial dynamics causes SN degeneration.