Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Jul 3.
Published in final edited form as: Arch Insect Biochem Physiol. 2022 Feb 1;110(1):e21867. doi: 10.1002/arch.21867

Probability that there is a mammalian counterpart of cardiac clock in insects

Sodikdjon A Kodirov 1,2,3
PMCID: PMC9250754  NIHMSID: NIHMS1816671  PMID: 35106839

Abstract

Whether or not the hyperpolarization-activated cyclic nucleotide-gated nonselective cation channel (HCN or funny current If) is involved in pacemaking – recurrent heartbeat, it is attributed to electrical activities in all excitable cells, including those of invertebrates. In latter group of animals prevailingly the electrical signals and function of heart in terms of chrono- and inotropy are elucidated. Although in simpler models including insects experimental outcomes are reproducible and robust, involvement of “cardiac clock” mechanism in pacemaking is not conclusive. In this assay, the mechanisms of heartbeat are synthesized by focused comparisons between insect and mammalian hearts.

Keywords: HCN, Heart rate, If channel

…invertebrate researchers face the added challenge of integrating data from different phyla, some of which have been separated from each other for more than 550 million years…

(Tierney, 2001).

1 |. INTRODUCTION

Physiological properties of organs in invertebrates are frequently compared with mammals, as the final aim is to gain a translation knowledge that could be applicable to humans (Nóblega et al., 2006; Pieńkowska et al., 2014; Renwrantz & Spielvogel, 2011). The foremost compared organs are the brain and heart (Kotsyuba & Dyachuk, 2021; S.-Rozsa et al., 1973). Also the majority of mammalian ion channel homologs including the ankyrin-transient receptor potential type A receptor are expressed in invertebrates (Kiss, 2003; Kostyuk et al., 1975; Marguerite et al., 2021).

One of the earlier recognized phenomenon is the spontaneous pacemaker activity of the heart derived by the oscillations of membrane potential (MP) (Tsien et al., 1979). Therefore, in mammals, two nonexclusive mechanisms were suggested and subsequently termed as internal (cytosol) and surface membrane oscillators. Later the same mechanisms were termed as intracellular Ca2+ and membrane voltage clocks, respectively (Lakatta & DiFrancesco, 2009).

The heart rate (HR) in invertebrates is modulated by three main mechanisms. Earlier recognized one occurs via neurohumoral factors – neurotransmitters, which are released postsynaptically into the myocardium and exert similar effects also when probed in vitro (Hill & Yantorno, 1979; Welsh, 1957; Zhuravlev, 1999). The second mechanism concerns hormones that are released into cardiovascular system by neurosecretory cells. Thus, the released hormones circulate and reach several organs including the heart. The release is a transient phenomenon and occurs after an action potential (AP) of neurons within the central nervous system (CNS) of insects (Ichikawa & Okada, 2002). The cluster of neurosecretory white neurons in molluscan CNS contain electron-dense granules and the “intensity of cell whiteness” correlates with excitability (Chase & Goodman, 1977). The number and size of neurosecretory neurons are age-dependent and the cluster organization is similar to those occurring in dedicated nuclei of mammals (Kojima et al. 2012; Kruatrachue et al., 1994; Potapenko et al., 2011). Although not extensively studied, there are granule cells adjacent to and within the heart tissue, which contain cardioactive peptides (Kodirov, 2011). The third mechanism is executed by the heart itself after a cardiac stretch. Therefore, the latter is not autonomous and occurs after the first or second or both mechanisms. However, this mechanism was evidenced considerably earlier, particularly in crustacean and molluscan hearts (Carlson, 1906). Importantly, this mechanism as assessed experimentally by mechanical tension does not restore the HR to levels observed in intact animals.

The heart of insects is of tubular origin as is that of human during early embryonic states (Battista et al., 2017). Therefore, the functioning of the heart could underlie a pumping either by dynamic suction or peristaltic motion. The latter two modes are true for human embryos in the absence of valves. In Drosophila the myocardium is constituted by only 52 pairs of cardiomyocytes, which are aligned along the endothelial cells in a lamina (Lalevée et al., 2006). In insects, despite a simpler heart, there is a circulation of hemolymph, but the cardiovascular system is not comprised of closed loop. However, the open-loop cardiovascular system in insects is relatively complex, as it involves alternations in flow directions and is governed by recurrent anterograde and retrograde heartbeats of distinct frequencies (Ichikawa & Okada, 2002).

In insects even allometric relationship between body mass (BM) and HR are examined, for example, in cockroach, Blaberus discoidalis (Birchard & Arendse, 2001), to compare with that of mammals. However, despite similarity – decrease in HR and increase in BM – the relationship did not exhibit a strict linear dependency. Nonlinear relationship is documented also for chameleon, Chamaeleo calyptratus, during embryogenesis in ovo (Nechaeva et al., 2005).

Nevertheless, the myogenic heart rhythm is influenced by several factors, while determinatory ones are the mechanisms by which the intracellular Ca2+ and pacemaking are handled by channels, exchangers, and transporters of cell membrane and cytoplasmic organelles (see further).

2 |. MODULATION OF HR

The heart of insects is simpler, but the basics of functioning are similar to mammals. In insects, a portion of muscular tissue tube is recognized as a true heart (Figure 1) because of the larger diameter and occurrences of rhythmic electrical activities in contrast to the narrower aorta, which supplies the head with hemolymph (Marguerite et al., 2021). However, the aorta also contracts and similar to the true heart consists of a striated muscle monolayer as described elsewhere (Papaefthimiou et al., 2002).

FIGURE 1.

FIGURE 1

Open in a new tab

Heart of insects. (a) Appearance of tube-shaped heart of Drosophila white pupa as visualized with GFP in vivo. Aorta is thinner, but the transition starts gradually from posterior part of true heart toward the anterior one. Two tracheas are located in parallel. (b) Heart of late third instar Drosophila larva after hybridization in situ revealing expression of Ork1 gene that encodes K2P – a two-pore domain K+ channel. Expression levels exhibit selectivity toward the cardiomyocytes of true heart. DAPI staining also has distinct patterns in both aorta and heart, which may hint that cell morphologies differ. Within the aorta there is a degree of parallelism among stained nuclei, while some of pairs are distanced also similarly. Modified (Lalevée et al., 2006). DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein

Environmental factors, for example, temperature may influence the frequency of heartbeats. The HR transiently increases to approximately 250 versus 150 beats · min−1 upon relocation of naïve larvae of Drosophila melanogaster to 37 versus 21°C (Marguerite et al., 2021). The latter larvae (UAS-TrpA1 – transient receptor potential type A receptor) expressed the native levels of channel and served as a control group. When animals with high expression of the TrpA1 receptors were subjected to the same procedure, the heartbeat reversibly ceased at 37°C. The HR of intact third instar D. melanogaster larvae was determined to be 2.83 Hz with new software (Zabihihesari et al., 2021). The estimation is derived from the most frequently contracting portion of tube that is presumably the true heart.

The heartbeat is sensitive also to venom. Although, in a preliminary study, the HR of Tenebrio molitor is increased marginally by approximately 6% in contrast to the stated 9% after application of 100 μM melittin polypeptide, a constituent of honeybee venom (Lubawy et al., 2019). The 9% is the upper range of SEM. Thus, the estimated EC50 is inadequate, since is based on a single data point. Also even addition of 10 μl physiological solution into the identical perfusate decreased the HR by approximately 4% from baseline 86 beats · min−1. The latter frequency consistently matches the 89 beats · min−1 observed during 5 h. The minimal observed mean frequency is 0.6 Hz (Papaefthimiou et al., 2002). The HR of T. molitor was decreased by herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), but at high 1 mM concentration compared to honeybee Apis mellifera macedonica, which was attributed to the absence of gap junction hemi-channels in former. In contrast to the statement, in latter animal 9 μM did not obliterated heartbeats, but deceased it by approximately 80% on average. Thus, the dose–response curve is unprecise for all three groups including the frog Rana ridibunda, as the maximum range - 100% is not reached.

Also neurotransmitters and related peptides may influence the heart performance and their repertoire is evolutionary conserved (Springer et al., 2004). In cockroach Nauphoeta cinerea the OA – octopamine at 15 μg/100 μl was concluded to increase the HR (Borges et al., 2020), but the difference between 103 versus 98 beats · min−1 of control is marginal. Especially, since the latter frequency is a baseline value for all three groups. Two mean data points during baseline and one immediately after are identical for control and OA groups in Figure 3b, which is unexpected. The latter citation in italic is used to distinguish from those in this study referred as to Figure 1 and others. The same approach applies also as to tables.

FIGURE 3.

FIGURE 3

Open in a new tab

Predicted wash-out effects. (a) Same as in Figure 2. Part of which was cut and pasted into (b) to simulate reversibility of heartbeats obliteration by Cd2+ exposure. To convince the originality of recording a small “nose” was added to the beginning of trace (+). The logic of physiology – a slow recovery of heart rate is substantiated by horizontal stretching. (c) Identical trace from (b) after careful horizontal compression. (d) A portion of (a) after stretching. Highlighted (Feliciano et al., 2011)

A selective antagonist of octopaminergic receptors phentolamine decreased the HR within approximately 2 min to 72 beats · min−1 (Borges et al., 2020). These opposite effects of agonist and antagonist hardly explain the involvement of the same underlying mechanism, even when both were applied concurrently. Effects of CEMR –crude extract of Manilkara rufula leaves (Caatinga tree) were of negative chronotropic nature and occurred faster (indication of wash-in start is not precise in Figure 3a). Effects are not strictly dose-dependent, as in contrary, it is higher with 50 versus 100 μg/100 μl and decline similarly. Interestingly, those of lowest concentration at 25 μg/100 μl do not decline.

The crustacean cardioactive peptide (CCAP) is present in CNS of invertebrates and have been extracted from whole heads of insect T. molitor (Furuya et al., 1993). However, its content in the brain is low, as “one head equivalent” of extracted CCAP exhibit almost identical effects with those of 10 fM of synthetic one on semi-isolated heart of tobacco hornworm Manduca sexta. Also 100 pM proctolin accelerates the HR in coleopteran insects including T. molitor, but effects saturate at approximately 70% by 10 nM (Sliwowska et al., 2001).

The HR modulation after neurosecretion by neurons of CNS is found also in insects, which e.g. are termed as bombyxin-producing (BP) cells in silkmoth Bombyx mori (Ichikawa & Okada, 2002). Since the frequency of neuronal APs not only decline, but may reach the 0 Hz as soon as the anterograde heartbeat starts, there might be a feedback loop between CNS and heart, or BP neurons simply are periodically oscillating neurons (PON) as in mollusks, albeit slower cycles. Therefore, alternations in slow HR – retrograde and fast HR – anterograde could be simply preprogrammed for heart to change the direction of hemolymph flow. Interestingly, during reverse–anterograde heartbeats the BP neurons transit into inactive state, that is, do not release bombyxin – insulin-related neuropeptide. The ultradian (more frequent cycles in contrast to circadian and infradian periods) bursting rhythm of B. mori BP neuron occurs heterogeneously and fastest interval has been 25 min, while anterograde heartbeats are observed during 6.5 min on average. In 1 day old beetle Zophobas atratus the retrograde and anterograde phases occur with shorter both durations and intervals (Lubawy et al., 2019). However, the HR during retrograde phase is consistently lower in both species, that is, about 12 versus 23 beats · min−1 of retrograde phase.

Although cardiac output, diastolic and systolic diameters, and stroke volume have increased linearly with body mass – exhibited allometric relationship, but the HR did not in B. discoidalis (Birchard & Arendse, 2001). Despite similitude to mammals – decrease in HR and increase in BM, the relationship did not exhibit a strict linear dependency as mentioned earlier. Moreover, the “linear model is questionable” because the R2 value was low at 0.5, though p < .0001. Besides, the diastolic and systolic diameters of heart obeyed the allometric relationship with R2 of 0.89 versus 0.84, respectively. In chameleon, Chamaeleo calyptratus, even a stable HR at approximately 69 beats · min−1 is documented during embryogenesis in ovo (Nechaeva et al., 2005).

The HR of insects is sensitive also to anesthesia. In D. melanogaster larvae the heartbeat is reversibly ceased by anesthetic tricaine mesylate (or MS-222), which apparently inhibits Nav channels in neurons of CNS (Stanley et al., 2020).

3 |. CARDIAC CLOCK

Even if a “cardiac clock” exists as a true pacemaker of heart, it should involve several organelles and ion channels within the cell and its membrane (Lakatta & DiFrancesco, 2009). These are Na+ and Ca2+ exchanger (NCX), ryanodine receptors (RyR), and sarcoplasmic and endoplasmic reticulum Ca2+–ATPase (SERCA). To some extent the latter may include also the Na,K-ATPase, when coupled with the functional abnormalities of endoplasmic reticulum (ER) (Kryvenko et al., 2021).

More complex is the organism, more elements would be required. A synergy among all these targets is the most plausible mechanism, though the crucial effect occurs via Ca2+ ions. Since also the intracellular Ca2+ is prone to rhythmicity during spontaneous release from internal stores, the latter was proposed to constitute Ca2+ clock. In contrast, the Ca2+ sources for AP initiation and maintenance of rhythm are available via cytoplasmic long lasting (L) and transient (T) types of Cav channels along the HCN (since non-selectively permeable also to Ca2+ ion) and a more precise Ca2+ cycler NCX. The latter could be unified under umbrella name of membrane clock. Because of potential dependency of aforementioned three types of channels, they are considered also as a voltage clock. Note that even a dynamic ion exchange between an organelle – sarcoplasmic reticulum and the cytosol alone already could be termed as SR clock, since it may greatly contribute to Ca2+ cycle. However, a coupling among all both minor and major mechanisms is a true driver of cardiac rhythm and is termed as a pacemaker clock (Lakatta & DiFrancesco, 2009).

The term clock and underlying system within the heart are perhaps derived from circadian brain rhythm triggered by neuronal clock or C–spikes, which are present also in insects (Moro & Huotari, 1998). The latter prompted to search for biological or even molecular clock in various experimental animals and their organs. Note that the circadian clock mechanism does not only involve dedicated receptors, but also interplay between those responsible for neurotransmission, for example, GABA and oxytocin (Chernysheva et al., 2013).

Even in earlier studies dedicated to mammalian sino-atrial node (SAN) one may encounter a skeptical tone in regard to three hypothetical sources of pacemaking (Satoh, 1995), namely the L-type inward calcium (ICa–L), outward potassium (Ik), and nonselective cationic currents termed as “funny” (If). Since the pacemaker hallmark – spontaneous diastolic depolarization starts at maximum diastolic potential (MDP) of −65 mV and terminates at the onset of AP at −60 mV, the involvements of ICa–L witha thresholdof activation at −40 mV is not expected. Therefore, the value of −40 mV corresponds to a holding potential during experiments with SAN cells in vitro. The same applies as to If via HCN, since the MP and timing of hyperpolarization exceeds that of diastolic depolarization (DD) range. The third source was proposed to be the IK, since its deactivation occurs around terminal repolarization phase of AP. However, activation of IK does not occur during DD. Therefore, additional sources were hypothesized and the role of T-type inward calcium current (ICa–T) was elucidated in SAN cardiomyocytes. Although, the baseline was stable, during application of 100 nM TTX the HR declined and steady-state level of inhibition was not reached during approximately 4 min. Therefore, subsequent gradual inhibitory effects during concurrent TTX and Ni2+ are hard to discern. Especially, the HR did not reverse, or even saturated. During the wash-out of both agents the amplitude of APs has increased, perhaps because of rebound effects after a long period of diminished rate. From superimposed pairs of APs it is not clear whether or not up to 100 μM Ni2+ does influence the amplitude. Even based on the activation range of ICa–T around −60 mV and effects of two selective blockers, Ni2+ and tetramethrin-induced HR decrease, no firm conclusion was drawn. Besides, there are other proteins, SERCA and RyR that along NCX trigger the cardiac excitation-contraction coupling. Finally, the Ca2+ itself at 10 mM and when applied concurrently with isoproterenol, obliterates the pacemaking of SAN myocytes (Satoh & Uchida, 1993).

It has been documented that the electrical activity and response of SAN myocytes are different, when tested is isolation or within the multi-cellular tissue, as Ca2+ antagonists diltiazem and D-600 do not influence the DD in the former case in contrast to latter (Koidl et al., 1986). An obliteration of APs by 1 μM D-600 is reversed by strong experimental hyperpolarization, which may explain cases, when no or minor effects are observed on spontaneous APs, as some compounds by acting via multiple targets, may also influence the resting membrane potential (RMP).

4 |. PACEMAKING VIA HCN

Whether or not the hyperpolarization-activated cyclic nucleotide-gated nonselective cation channel (HCN or If) is involved in pacemaking – recurrent heartbeat, it is attributed to electrical activities or membrane potential in all excitable cells. The underlying currents alone are unique, emerged as iK2 and referred subsequently as to ‘funny’ If conductance (DiFrancesco & Ohba, 1978; Noble & Tsien, 1968; Noma & Irisawa, 1976), but still more emphasis is centered on potential role of HCN in pacemaking (Bruzauskaite et al., 2016).

At least one study presents a contrary evidence in invertebrates, that is, excludes the involvement of HCN in HR modulation in insect mealworm beetle Tenebrio molitor (Feliciano et al., 2011). Since the HR was insensitive to CsCl and ZD7288. Although the ZD7288 is an established antagonist of If channel, the Cs+ may exert additional effects on Kv subunits.

The HR of T. molitor is often studied. Also arrhythmicity is present in beetles already at first week of life and its index during aging increases to 3.2 versus 0.2 (Pacholska-Bogalska et al., 2018). An increase in diastolic intervals may accompany this development. A round values of 40.0 ± 5.0 μm for heart diameter at diastolic state is also not expected despite the high sensitivity of frequency to solution flow and even if the “perfusion rates lower than 1 ml/min markedly reduced BR and led to rhythm irregularity, whereas little change was observed when flow rate was increased above 1 ml/min” (Feliciano et al., 2011). That is, the beating rate (BR) should be measured even not at 0.99 ml/min exchange tempo of perfusate.

There are no absolute specific or selective compounds that could target only HCN in more physiological settings, including the ivabradine. As if it “reduces heart rate by six beats/min surely does not mean that If is the primary pacemaker mechanism. Furthermore, the development of this drug was catalyzed by the sustained dogma that If current is the primary pacemaker mechanism” (Lakatta & DiFrancesco, 2009). The latter will not alter even if the ivabradine was tested on patients with a round number of 11,000 in 33 countries, since the impact of this selective HCN antagonist must be robust in vitro. Theoretically, indeed Δ6 could mean 54 versus 60 beats · min−1 of control and one have to consider also the natural heart rate variability (HRV). Therefore, the apogeal poetic beauty and scientific creativeness of acronym for a trial name as BEAUTIfUL is minimal, even for our century. Therefore, the HCN currents could not be considered funny anymore, but beautiful, as it is indeed for many scientist.

Also sevoflurane, a volatile anesthetic, at 440 μM concentration decelerated the frequency of spontaneous APs in isolated SAN cells to up 25%, while effects correlated not only with reduction of If, but also ICa–L,ICa–T, and IKs (Kojima et al., 2012). Note that the percentile of block was least for HCN at approximately 14 versus 30, 31% and 37% for other channels, respectively. Similar and concurrent effects have been proved by numerous solid a single author studies using natural and synthetic compounds, including Ginkgo biloba (extracts from the leaves are considered therapeutic supplement in regard to also anxiety and memory), caffeine, Ni2+, nicotine, ryanodine, taurine, and tetramethrin (Satoh, 1993, 1995, 1997a, 1997b, 1999, 2002; Satoh, 2005).

5 |. NEITHER CA2+ CLOCK NOR HCN IS A DOMINANT PACEMAKER

In mammals it is still not straight forward to decipher whether the pacemaker HCN channel, a Ca2+ and membrane clock, or all contributively regulate the HR (Lakatta & DiFrancesco, 2009). One of participating mechanisms for cardiac spontaneous rhythmicity may underlie corresponding orchestrated fluctuations in intracellular calcium concentration during release and uptake by organelles and constitute the Ca2+ clock. These fluctuations alone are not sufficient to drive the pacemaker rhythm of SAN, therefore a coordinated activity of Na–Ca exchanger (NCX) along the “classic sarcolemmal membrane voltage clock” are required. The membranous players that alter the MP of SAN cells are Cav, HCN, and Kv channels. Recently, the ITASK prior known as ILeak current and underlying K2P3.1 – a two-pore domain K+ channel was considered to influence the rhythm (Kodirov, under review).

In insects, it is possible that instead also these K channels play some role in pacemaking. As despite the presence of Kv with defined roles, also ORK1 – K2P that contributes K leak currents obliterates the heartbeat of Drosophila white pupae (Lalevée et al., 2006). However, the latter effect occurs only after additional exogenous ORK1 – UAS > Ork1 introduced by overexpression. Also it is unclear how the MDP of −30 mV was estimated in table 1 in the absence of AP – heartbeat. The RMP of UAS > Ork1 group correlated with the content of K+ yielding −76 mV at 5 mM, while −31 at 25 and −18 at 49, respectively. The RMP of −76 mV is without merit, although the value is derived from solid recordings after impaling with sharp electrodes and sufficient times to equilibrate, since the MDP for groups of control, gain, and loss of function mutations are very similar and low by ranging narrowly from −17 to −20 mV. It is inadequate to state “results not shown” within table 1 even if ‘there are no significant differences between yw;CS and 1029-Gal4 x yw;CS, which have been taken as control’ (Lalevée et al., 2006). As it is not clear either yw;CS or 1029-Gal4 x yw;CS is the main reference group. Note that the HR was not significantly apart in K2P6.3 and K2P7.1 mutants or control groups at 21°C. An increase of HR in mutants at higher temperatures is without crucial significance, as the control group was subjected only to 21°C.

As mentioned above, based on established pharmacology the HCN does not contribute to HR in T. molitor (Feliciano et al., 2011). Therefore, to use the latter against dogma (Kodirov, 2021), the article was evaluated again. However, the statistically relevant digits in values of 33.3 ± 3.3 versus 61.2 ± 2.9 beats · min–1 after application of 30 μM BHQ (2,5-tert-butyl-4,5 hydroquinone), a competitive inhibitor of the SERCA, prompted to scrutinize the visualized data.

These and similar digits were seen before, but were forced to trust and believe that they might have place in nature and science. How it is logical that the RR interval in ECG is apart at 106 versus 113 ms (Lubberding et al., 2020), but the PR and QRS are identical at respective 33 ± 1 and 11 ± 0.2 ms at the same time and conditions for two different groups, control – db/+ and diabetic – db/db mice in table 1? Are these digits, 216 ± 44 versus 260 ± 39% really not apart and statistically insignificant for db/+ versus db/db mice and carbachol effects on RR? In another set of data for db/db mice in Figure 4a the base RR was approximately 150 ms and 200 mg/kg isoprenaline reduced it to 90 ms. If the control value was 113 ms as mentioned above, then differences would be, highly likely, insignificant. Though, this notion is disproved by results in Figure 2f. The differences in base RR can not be attributed to concurrent monitoring of blood pressure (BP). Identical digits of 19 ± 1 and 7 ± 1 ms for standard deviation of RR (SDRR) intervals and root mean square of the successive differences (RMSSD) of RR interval appear also for different groups or conditions. In total, the possible role of SAN and non-involvement of baroreceptors are uncertain. Indeed, all depends on expectations, as approximately 16% BP lowering by isoprenaline could be considered for the absence of effects. The behavior of RR in response to atropine was similar, yet it obliterated the arrhythmia. Nevertheless, it is hard to follow the precise degree of significance among incidence of arrhythmia counts, that is approximately 16 versus 0 for db/+ or 64 versus absolute 0 for db/db mice. Would not be also differences between 1 versus 0 significant and to which degree?

FIGURE 4.

FIGURE 4

Open in a new tab

Simulation of contractions during drug wash-out. (a) Similar to Figure 3 a part of Figure 2 was cut, copied and pasted into (b) to convince reviewers and readers that the Cd2+ effects were not terminal. (c) Identical trace from (b) after horizontal compression in PowerPoint. (d) Initial portion of (a) after stretching to match (b). Note that in (c) and (d) a retrospect manipulation alters also the thickness of traces hinting that those in Figure 2 are performed by other methods, and directly in dedicated software. Highlighted (Feliciano et al., 2011)

FIGURE 2.

FIGURE 2

Open in a new tab

Electrical activity and contraction of the heart. (a) and (b) Electrogram and mechanical contraction under control conditions in the mealworm beetle, Tenebrio molitor. (c) and (d) Same preparation after exposure to Cd2+ at 100 μM concentration during external perfusion. (e) and (f) Imaginary wash-out effects (see Figures 3 and 4). Amplitude scale for (a), (c), (e) and arbitrary unit for (b), (d), (f), while time scale for all panels are identical. Modified (Feliciano et al., 2011)

Note that the aforementioned study was extensively reviewed leading to conclusion that the ‘heart was insensitive to carbamylcholine, CsCl, isoproterenol, norepinephrine, and ZD7288 in established concentrations. Since specifically ZD7288 did not affect the HR, there is either no dedicated pacemaker region in invertebrates or HCN channels are absent, or both are distinct compared to vertebrates’ (Kodirov et al., 2019). In particular, and in regard to HR – heart rate, the Figure 2 was covered entirely as ‘BDM almost obliterates the contraction within 10 min, but the electrical activity changes only in rate and amplitude (Feliciano et al., 2011). The depolarizing and repolarizing polarity distribution of electrograms also differ compared to control. However, 100 μM Cd2+ completely and reversibly diminishes both parameters’ (Kodirov et al., 2019). Regrettably, Figure 2b is based on cut, copy and paste approach along the horizontal stretching – “decreased rate” to mimic hypothetical insufficient wash-out effects (Figures 2–4). The latter is unnecessary, especially for invertebrates, as responses and signals in simpler animal models are reproducible and robust (Kodirov, 2011; Kodirov et al., 2021; S.-Rozsa, 1979; Zhuravlev et al., 2001).

Several studies relied on Feliciano et al. (2011) when it concerned the heart rhythm and cardioregulation (Chowański et al., 2016; Dos Santos et al., 2019; Pacholska-Bogalska et al., 2018). But major conclusions, namely similitude between insects and vertebrates in automatism and involvement of Cav channels, Na+/Ca2+ exchanger and SR Ca2+ release is without merit, especially, when reported changes in HR is derived from either experimental conditions or artifacts (Figures 3 and 4). Note that even in vertebrate heart cardiomyocyte the estimation of SR release of Ca2+ is not straight forward, as introducing 24 mM caffeine solution into the chamber dramatically alters the Fluo4 fluorescence signals, though cells were not present yet (Loescher et al., 2019).

Previously, it was stated that “the HR was Ca2+ and concentration-dependent, although the effects started to saturate around 4 mM” (Kodirov et al., 2019) in contrary to assumptive approximately 8 mM based on Figure 3 (Feliciano et al., 2011). As the 4 mM is a standard concentration and beforehand there are only 1 and 2 mM tested doses, the exponential increase in HR can not be substantiated precisely. Especially, when it is not clear from graph that whether or not the heart beats in 0 mM Ca2+ and n numbers are missing for all data points. The latter along the cadmium experiments (if those were genuine, at least partially) would substantiate that the electrical activity in heart of T. molitor is generated by L-type Cav channels.

It was also noticed the HR was similarly slowed to approximately 30 versus 60 beats min−1 in the presence of BHQ, caffeine, ryanodine, thapsigargin, or concurrent application of two latter substances. Thus, there is no selectivity or distinct affinity. If 100 μM thapsigargin exerts only negative chronotropic effects, why the diastolic diameter changes dramatically in Figure 4a? Note that opposite inotropic effects are stated for 5-HT. Effects of this classical neurotransmitter and Ca2+ in invertebrates are also not novel and has been substantiated at more precise levels including cardiac action potential duration (APD) and E–C coupling (Hill & Yantorno, 1979). Although the tested concentrations are often higher (Moulis & Huddart, 2006). Not only the cylindrical heart (Figure 5), but also hemocytes within the lumen and production of nitric oxide (NO) has been visualized in Vietnamese stick insect Baculum extradentatum (da Silva et al., 2012).

FIGURE 5.

FIGURE 5

Open in a new tab

Cylindrical heart of insects. (Left) scanning electron microscopy (SEM) revealing the morphologies of both heart and intimately attached lateral cardiac nerves. The latter is a hub for bipolar neurons. Modified (da Silva et al., 2011). This and next preparation represent part of heart of Vietnamese stick insect, Baculum extradentatum. (Right) Whole-mount preparation of heart. There are numerous hemocytes grouped in clusters. Detected NADPH-diaphorase activity in hemocytes substantiates that they are putative NO-releasing cells in insects. Highlighted (da Silva et al., 2012)

The ZD7288 effects on HCN depends on application time, but the used concentration at 5 versus 100 μM might be lower compared to relevant studies (Feliciano et al., 2011; Tompkins et al., 2009). Though the concentration of Cs+ used to block HCN was higher at 10 versus 2 mM. Application time of 2, 4, or even 15 min might not be sufficient to exert effects, since in this preparation the lumen of heart is not directly exposed to drugs or ZD7288. Besides, Cd2+ at 100 μM or higher concentration may also inhibit HCN channels as shown for its counterpart IAB in crayfish Procambarus clarkia (Araque et al., 1995).

Since the cadmium effects was the second important observation and that of serotonin more robust (Feliciano et al., 2011), the merit of Ca2+ that is possibly released by SR is insufficient. Besides, in mammals the latter is not a dominant factor in determining the pacemaker role of most dedicated region – sinoatrial node (Honjo et al., 2003). However, indisputable is the role of membranous Ca2+ flow in SAN cells that generate AP upstroke (Denyer & Brown, 1990). Despite the presence of Nav in SAN, the amplitude and rate of spontaneous APs are only marginally decreased by heroic concentration of TTX at 30 μM.

6 |. CONCLUSIONS

Summa summarum and based on visualized experimental shortcomings neither funny currents nor Ca2+ modulate the pacemaker heart rate of insect T. molitor (Feliciano et al., 2011). Although, the concept of beating clock at pacemaker rate is plausible, but when it involves several elements and most probably simultaneously, its fate is not tractable, at least not in vitro. Therefore, questions arise as to their percentile contributions to HR and roles as prevailingly dominance. Insects do not have SAN, although they possess cardiac myocytes that generate rhythms. In many crustaceans, cardiac rhythm is generated by a small network of neurons residing in the heart (Kodirov et al., 2019). In vertebrates SAN myocytes are drivers of HR among all cardiac cells. The same needs to be defined for a single SAN pacemaker cell in regard to all members of cardiac clock. Until then it will remain unclear which factor or clock is a driver – pacemaker and not a follower – pacetaker (Lakatta & DiFrancesco, 2009).

ACKNOWLEDGMENTS

I am grateful to my mentor Vladimir Leonidovich Zhuravlev 71 (Leningrad University, ROC) for determining my path in science with research on CNS and heart of Giant African snail Achatina fulica F. and to Dr. Clara Downey (University of Texas) for critical reading of earlier version. The study was partially supported by a grant 8809 from the Ministry of Education and Science, Federal target program “Research and Pedagogical Cadre for Innovative RUSSIA” and National Institute of Health, USA.

Funding information

Ministry of Education and Science of the Russian Federation; National Institutes of Health, USA

Footnotes

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

REFERENCES

  1. Araque A, Cattaert D, & Buño W (1995). Cd2+ regulation of the hyperpolarization-activated current IAB in crayfish muscle. Journal of General Physiology, 105, 725–744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Battista NA, Lane AN, & Miller LA (2017). On the dynamic suction pumping of blood cells in tubular hearts. In Layton A, & Miller L (Eds.), Women in mathematical biology (pp. 211–231). Springer. [Google Scholar]
  3. Birchard GF, & Arendse AU (2001). An allometric analysis of oxygen consumption rate and cardiovascular function in the cockroach, Blaberus discoidalis. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology, 129, 339–344. [DOI] [PubMed] [Google Scholar]
  4. Borges BT, de Brum Vieira P, Leal AP, Karnopp E, Ogata BAB, Rosa ME, Barreto YC, Oliveira RS, Belo CAD, & Vinadé L (2020). Modulation of octopaminergic and cholinergic pathways induced by Caatinga tree Manilkara rufula chemical compounds in Nauphoeta cinerea cockroaches. Pesticide Biochemistry and Physiology, 169, 104651. [DOI] [PubMed] [Google Scholar]
  5. Bruzauskaite I, Bironaite D, Bagdonas E, Skeberdis VA, Denkovskij J, Tamulevicius T, Uvarovas V, & Bernotiene E (2016). Relevance of HCN2-expressing human mesenchymal stem cells for the generation of biological pacemakers. Stem Cell Research & Therapy, 7, 67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carlson AJ (1906). Comparative physiology of the invertebrate heart. V. The heart rhythm under normal and experimental conditions. American Journal of Physiology, 16,47–66. [Google Scholar]
  7. Chase R, & Goodman HE (1977). Homologous neurosecretory cell groups in the land snail Achatina fulica and the sea slug Aplysia californica. Cell and Tissue Research, 176, 109–120. [DOI] [PubMed] [Google Scholar]
  8. Chernysheva MP, Romanova IV, & Mikhrina AL (2013). Effect of retinol on interaction of the protein period 1, oxytocin, and GABA at the prenatal period of formation of the circadian clock-mechanism in rats. Journal of Evolutionary Biochemistry and Physiology, 49,97–104. [PubMed] [Google Scholar]
  9. Chowański S, Lubawy J, Urbański A, & Rosiński G (2016). Cardioregulatory functions of neuropeptides and peptide hormones in insects. Protein & Peptide Letters, 23, 913–931. [DOI] [PubMed] [Google Scholar]
  10. Dos Santos DS, Zanatta AP, Martinelli AHS, Rosa ME, de Oliveira RS, Pinto PM, Peigneur S, Tytgat J, Orchard I, Lange AB, & Carlini CR, Dal Belo CA (2019). Jaburetox, a natural insecticide derived from Jack Bean Urease, activates voltage-gated sodium channels to modulate insect behavior. Pesticide Biochemistry and Physiology, 153, 67–76. [DOI] [PubMed] [Google Scholar]
  11. da Silva R, da Silva SR, & Lange AB (2012). The regulation of cardiac activity by nitric oxide (NO) in the Vietnamese stick insect, Baculum extradentatum. Cellular Signalling, 24, 1344–1350. [DOI] [PubMed] [Google Scholar]
  12. da Silva R, da Silva SR, & Lange AB (2011). Effects of crustacean cardioactive peptide on the hearts of two Orthopteran insects, and the demonstration of a Frank-Starling-like effect. General and Comparative Endocrinology, 171, 218–224. [DOI] [PubMed] [Google Scholar]
  13. Denyer JC, & Brown HF (1990). Rabbit sino-atrial node cells: Isolation and electrophysiological properties. Journal of Physiology, 428, 405–424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. DiFrancesco D, & Ohba M (1978). Dependence of the apparent reversal potential for the pace-maker current iK2 on its degree of activation in cardiac Purkinje fibres. Journal of Physiology, 280, 73–74. [PubMed] [Google Scholar]
  15. Feliciano DF, Bassani RA, Oliveira PX, & Bassani JWM (2011). Pacemaker activity in the insect (T. molitor) heart: Role of the sarcoplasmic reticulum. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 301, R1838–R1845. [DOI] [PubMed] [Google Scholar]
  16. Furuya K, Liao S, Reynolds SE, Ota RB, Hackett M, & Schooley DA (1993). Isolation and identification of a cardioactive peptide from Tenebrio molitor and Spodoptera eridania. Biological Chemistry Hoppe-Seyler, 374, 1065–1074. [DOI] [PubMed] [Google Scholar]
  17. Hill RB, & Yantorno RE (1979). Inotropism and contracture of Aplysiid ventricles as related to the action of neurohumors on resting and action potentials of molluscan hearts. American Zoologist, 19, 145–162. [Google Scholar]
  18. Honjo H, Inada S, Lancaster MK, Yamamoto M, Niwa R, Jones SA, Shibata N, Mitsui K, Horiuchi T, Kamiya K, Kodama I, & Boyett MR (2003). Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity. Circulation Research, 92, e41–e44. [DOI] [PubMed] [Google Scholar]
  19. Ichikawa T, & Okada Y (2002). Co-ordination of firing activity of neurosecretory cells with cardiac activity in the silkmoth Bombyx mori. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology, 133, 117–124. [DOI] [PubMed] [Google Scholar]
  20. Kiss T (2003). Evidence for a persistent Na-conductance in identified command neurones of the snail, Helix pomatia. Brain Research, 989,16–25. [DOI] [PubMed] [Google Scholar]
  21. Kodirov SA (2011). The neuronal control of cardiac functions in Molluscs. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology, 160, 102–116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kodirov SA (2021). HCN and nicotine. Acta Physiol, 232, e13651. [DOI] [PubMed] [Google Scholar]
  23. Kodirov SA, Brachmann J, Safonova TA, & Zhuravlev VL (2021). Inactivation of native K channels. Journal of Membrane Biology,. 10.1007/s00232-021-00195-w [DOI] [PubMed] [Google Scholar]
  24. Kodirov SA, Psyrakis D, Brachmann J, & Zhuravlev VL (2019). Limulus and heart rhythm. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology, 331, 61–79. [DOI] [PubMed] [Google Scholar]
  25. Koidl B, Tritthart HA, & MacLeod RS (1986). Different effects of calcium-antagonists on automaticity in single pacemaker cells and in synchronized networks of cultured embryonic heart muscle cells. Journal of Molecular and Cellular Cardiology, 18, 207–217. [DOI] [PubMed] [Google Scholar]
  26. Kojima A, Kitagawa H, Omatsu-Kanbe M, Matsuura H, & Nosaka S (2012). Inhibitory effects of sevoflurane on pacemaking activity of sinoatrial node cells in guinea-pig heart. British Journal of Pharmacology, 166(7), 2117–2135. 10.1111/j.1476-5381.2012.01914.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kostyuk PG, Krishtal OA, & Pidoplichko VI (1975). Effect of internal fluoride and phosphate on membrane currents during intracellular dialysis of nerve cells. Nature, 257, 691–693. [DOI] [PubMed] [Google Scholar]
  28. Kotsyuba E, & Dyachuk V (2021). Localization of neurons expressing choline acetyltransferase, serotonin and/or FMRFamide in the central nervous system of the decapod shore crab Hemigrapsus sanguineus. Cell and Tissue Research, 383, 959–977. [DOI] [PubMed] [Google Scholar]
  29. Kruatrachue M, Seehabutr V, Chavadej J, Sretarugsa P, Upatham ES, & Sobhon P (1994). Development and histological characteristics of neurosecretory cells in the cerebral ganglia of Achatina fulica (Bowdich). Moll Res, 15, 29–37. [Google Scholar]
  30. Kryvenko V, Vagin O, Dada LA, Sznajder JI, & Vadász I (2021). Maturation of the Na, K-ATPase in the endoplasmic reticulum in health and disease. Journal of Membrane Biology, 254, 447–457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lakatta EG, & DiFrancesco D (2009). What keeps us ticking: A funny current, a calcium clock, or both? Journal of Molecular and Cellular Cardiology, 47, 157–170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lalevée N, Monier B, Sénatore S, Perrin L, & Sémériva M (2006). Control of cardiac rhythm by ORK1, A Drosophila two-pore domain potassium channel. Current Biology, 16, 1502–1508. [DOI] [PubMed] [Google Scholar]
  33. Loescher CM, Gibson LM, & Stephenson DG (2019). Dantrolene sodium increases calcium binding by human recombinant cardiac calsequestrin and calcium loading by sheep cardiac sarcoplasmic reticulum. Acta Physiologica (Oxford, England), 226, e13261. [DOI] [PubMed] [Google Scholar]
  34. Lubawy J, Urbanski A, Mrówczyńska L, Matuszewska E, Światły-Błaszkiewicz A, Matysiak J, & Rosinski G (2019). The influence of bee venom melittin on the functioning of the immune system and the contractile activity of the insect heart. A preliminary study. Toxins, 11, 494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lubberding AF, Pereira L, Xue J, Gottlieb LA, Matchkov VV, Gomez AM, & Thomsen MB (2020). Aberrant sinus node firing during β2-adrenergic stimulation leads to cardiac arrhythmias in diabetic mice. Acta Physiologica (Oxford, England), 229, e13444. [DOI] [PubMed] [Google Scholar]
  36. Marguerite NT, Bernard J, Harrison DA, Harris D, & Cooper RL (2021). Effect of temperature on heart rate for Lucilia sericata (syn Phaenicia sericata) and Drosophila melanogaster with altered expression of the TrpA1 receptors. Insects, 12, 38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Moro SD, & Huotari M (1998). Clock-spiking cells not only in the eye of the fly, but also in the antenna! Acta Neurobiologiae Experimentalis, 58, 277–281. [DOI] [PubMed] [Google Scholar]
  38. Moulis A, & Huddart H (2006). RFamide neuropeptide actions on the molluscan ventricle: Interactions with primary neurotransmitters. Comparative biochemistry and physiology. Toxicology & pharmacology: CBP, 142, 95–102. [DOI] [PubMed] [Google Scholar]
  39. Nechaeva MV, Makarenko IG, Tsitrin EB, & Zhdanova NP (2005). Physiological and morphological characteristics of the rhythmic contractions of the amnion in veiled chameleon (Chamaeleo calyptratus) embryogenesis. Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology, 140, 19–28. [DOI] [PubMed] [Google Scholar]
  40. Noble D, & Tsien RW (1968). The kinetics and rectifier properties of the slow potassium current in cardiac Purkinje fibres. Journal of Physiology, 195, 185–214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nóblega HG, Rigon F, Stenert C, Faccioni-Heuser MC, & Achaval M (2006). Permeability of the haemolymph-neural interface in the terrestrial snail Megalobulimus abbreviatus (Gastropoda, Pulmonata): An ultrastructural approach. Comp. Biochem. Physiol. A Mol Integ Physiol, 144, 119–124. [DOI] [PubMed] [Google Scholar]
  42. Noma A, & Irisawa H (1976). Membrane currents in the rabbit sinoatrial node cell as studied by the double microelectrode method. Pflugers Archiv, 364, 45–52. [DOI] [PubMed] [Google Scholar]
  43. Pacholska-Bogalska J, Szymczak M, Marciniak P, Walkowiak-Nowicka K, & Rosiński G (2018). Heart mechanical and hemodynamic parameters of a beetle, Tenebrio molitor, at selected ages. Archives of Insect Biochemistry and Physiology, 99, e21474. [DOI] [PubMed] [Google Scholar]
  44. Papaefthimiou C, Pavlidou V, Gregorc A, & Theophilidis G (2002). The action of 2,4-dichlorophenoxyacetic acid on the isolated heart of insect and amphibia. Environmental Toxicology and Pharmacology, 11, 127–140. [DOI] [PubMed] [Google Scholar]
  45. Pienkowska JR, Kosicka E, Wojtkowska M, Kmita H, & Lesicki A (2014). Molecular identification of first putative aquaporins in snails. Journal of Membrane Biology, 247, 239–252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Potapenko ES, Biancardi VC, Florschutz RM, Ryu PD, & Stern JE (2011). Inhibitory-excitatory synaptic balance is shifted toward increased excitation in magnocellular neurosecretory cells of heart failure rats. Journal of Neurophysiology, 106, 1545–1557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Renwrantz L, & Spielvogel F (2011). Heart rate and hemocyte number as stress indicators in disturbed hibernating vineyard snails, Helix pomatia. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 160, 467–473. [DOI] [PubMed] [Google Scholar]
  48. Satoh H (1993). Positive and negative chronotropic effects of caffeine in spontaneously beating rabbit sino-atrial node cells. General Pharmacology, 24, 1223–1230. [DOI] [PubMed] [Google Scholar]
  49. Satoh H (1995). Role of T-type Ca2+ channel inhibitors in the pacemaker depolarization in rabbit sino-atrial nodal cells. General Pharmacology, 26, 581–587. [DOI] [PubMed] [Google Scholar]
  50. Satoh H (1997a). Effects of nicotine on spontaneous activity and underlying ionic currents in rabbit sinoatrial nodal cells. General Pharmacology, 28, 39–44. [DOI] [PubMed] [Google Scholar]
  51. Satoh H (1997b). Electrophysiological actions of ryanodine on single rabbit sinoatrial nodal cells. General Pharmacology, 28, 31–38. [DOI] [PubMed] [Google Scholar]
  52. Satoh H (1999). Taurine modulates IKr but IKs in guinea-pig ventricular cardiomyocytes. British Journal of Pharmacology, 126,87–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Satoh H (2002). Modulation by nicotine of the ionic currents in guinea pig ventricular cardiomyocytes. Relatively higher sensitivity to IKr and IK1. Vascular pharmacology, 39, 55–61. [DOI] [PubMed] [Google Scholar]
  54. Satoh H (2005). Suppression of pacemaker activity by Ginkgo biloba extract and its main constituent, bilobalide in rat sino-atrial nodal cells. Life Sciences, 78, 67–73. [DOI] [PubMed] [Google Scholar]
  55. Satoh H, & Uchida T (1993). Morphological and electrophysiological changes induced by calcium ionophores (A23187 and X-537A) in spontaneously beating rabbit sino-atrial node cells. General Pharmacology, 24,49–57. [DOI] [PubMed] [Google Scholar]
  56. Sliwowska J, Rosinski G, Nässel DR (2001). Cardioacceleratory action of tachykinin-related neuropeptides and proctolin in two coleopteran insect species. Peptides, 22(2), 209–217. 10.1016/s0196-9781(00)00384-3 [DOI] [PubMed] [Google Scholar]
  57. Springer J, Ruth P, Beuerlein K, Westermann B, & Schipp R (2004). Immunohistochemical localization of cardio-active neuropeptides in the heart of a living fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata). Journal of Molecular Histology, 35, 21–28. [DOI] [PubMed] [Google Scholar]
  58. Rozsa S, K. (1979). Heart regulatory neural network in the central nervous system of Achatina fulica (Ferussac) (Gastropoda, Pulmonata). Comparative Biochemistry and Physiology, 63, 435–445. [Google Scholar]
  59. S.-Rozsa K, Kiss T, & Szoke V-I. (1973). On the role of bioactive substances in the rhythm regulation of the heart muscle cells of Gastropoda and Insecta. In Salanki J (Ed.), Neurobiology of invertebrates. Mechanisms of rhythm regulation (pp. 167–181). Akademiai Kiado. [Google Scholar]
  60. Stanley CE, Adams R, Nadolski J, Amrit E, Barrett M, Bohnett C, Campbell K, Deweese K, Dhar S, Gillis B, Hill C, Inks M, Kozak K, Larson A, Murtaza I, Nichols D, Roberts R, Tyger H, Waterbury C, & Cooper RL (2020). The effects of tricaine mesylate on arthropods: Crayfish, crab and Drosophila. Invertebrate Neuroscience, 20, 10. [DOI] [PubMed] [Google Scholar]
  61. Tierney AJ (2001). Structure and function of invertebrate 5-HT receptors: A review. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 128, 791–804. [DOI] [PubMed] [Google Scholar]
  62. Tompkins JD, Lawrence YT, & Parsons RL (2009). Enhancement of Ih, but not inhibition of IM, is a key mechanism underlying the PACAP-induced increase in excitability of guinea pig intrinsic cardiac neurons. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 297, R52–R59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Tsien RW, Kass RS, & Weingart R (1979). Cellular and subcellular mechanisms of cardiac pacemaker oscillations. Journal of Experimental Biology, 81, 205–215. [DOI] [PubMed] [Google Scholar]
  64. Welsh JH (1957). Serotonin as a possible neurohumoral agent: Evidence obtained in lower animals. Annals of the New York Academy of Sciences, 66, 618–630. [DOI] [PubMed] [Google Scholar]
  65. Zabihihesari A, Khalili A, Hilliker AJ, & Rezai P (2021). Open access tool and microfluidic devices for phenotypic quantification of heart function of intact fruit fly and zebrafish larvae. Computers in Biology and Medicine, 132, 104314. [DOI] [PubMed] [Google Scholar]
  66. Zhuravlev V, Bugaj V, Kodirov SA, Safonova T, & Staruschenko A (2001). Giant multimodal heart motoneurons of Achatina fulica: A new cardioregulatory input in pulmonates. Comp Biochem Physiol Part A, 130, 183–196. [DOI] [PubMed] [Google Scholar]
  67. Zhuravlev VL (1999). Mechanisms of neurohumoral control of the heart in gastropods. Journal of Evolutionary Biochemistry and Physiology, 35,85–103. [Google Scholar]

RESOURCES