Larval Heart Research Articles

Effects of ocean acidification on early life-history stages of the intertidal porcelain crab Petrolisthes cinctipes

Intertidal zone organisms naturally experience daily fluctuations in pH, presently reaching values beyond what is predicted for open ocean surface waters from ocean acidification (OA) by the year 2100, and thus present an opportunity to study the pH sensitivity of organisms that are presumably adapted to an acidified environment. The intertidal zone porcelain crab, Petrolisthes cinctipes, was used to study physiological responses to low pH in embryonic, larval and newly recruited juvenile life-history stages. In these crabs, embryonic development occurs in the pH-variable intertidal zone (pH 6.9-9.5), larvae mature in the more stable pelagic environment (pH 7.9-8.2), and juvenile crabs settle back into the pH-variable intertidal zone. We examined survival, cardiac performance, energetics and morphology in embryonic, larval and juvenile crabs exposed to two pH conditions (pH 7.9 and 7.6). Embryos and larvae were split by brood between the pH treatments for 9 days to examine brood-specific responses to low pH. Hatching success did not differ between pH conditions, but ranged from 30% to 95% among broods. Larval survival was not affected by acidification, but juvenile survival was reduced by ~30% after longer (40 days) exposure to low pH. Embryonic and larval heart rates were 37% and 20% lower at low pH, and there was a brood-specific response in embryos. Embryos did not increase in volume under acidified conditions, compared with a 15% increase in ambient conditions. We conclude that sustained exposure to low pH could be detrimental to P. cinctipes embryos and larvae despite the fact that embryos are regularly exposed to naturally fluctuating hypercapnic water in the intertidal zone. Importantly, our results indicate that early life-history stage responses to OA may be brood specific through as yet undetermined mechanisms.

Analysis of postembryonic heart development and maturation in the zebrafish, Danio rerio

Cardiac maturation is vital for animal survival and must occur throughout the animal's life. Zebrafish are increasingly used to model cardiac disease; however, little is known about how the cardiovascular system matures. We conducted a systematic analysis of cardiac maturation from larvae through to adulthood and assessed cardiac features influenced by genetic and environmental factors. We identified a novel step in cardiac maturation, termed cardiac rotation, where the larval heart rotates into its final orientation within the thoracic cavity with the atrium placed behind the ventricle. This rotation is followed by linear ventricle growth and an increase in the angle between bulbous arteriosus and the ventricle. The ventricle transitions from a rectangle, to a triangle and ultimately a sphere that is significantly enveloped by the atrium. In addition, trabeculae are similarly patterned in the zebrafish and humans, both with muscular fingerlike projections and muscle bands that span the cardiac chamber. Of interest, partial loss of atrial contraction in myosin heavy chain 6 (myh6/wea(hu423/+)) mutants result in the adult maintaining a larval cardiac form. These findings serve as a foundation for the study of defects in cardiovascular development from both genetic and environmental factors.

Development of cardiovascular function in the marine gastropodLittorina obtusata(Linnaeus)

The molluscan cardiovascular system typically incorporates a transient extracardiac structure, the larval heart, early in development, but the functional importance of this structure is unclear. We documented the ontogeny and regulatory ability of the larval heart in relation to two other circulatory structures, the true heart and the velum, in the intertidal gastropod Littorina obtusata. There was a mismatch between the appearance of the larval heart and the velum. Velar lobes appeared early in development (day 4), but the larval heart did not begin beating until day 13. The beating of the larval heart reached a maximum on day 17 and then decreased until the structure itself disappeared (day 24). The true heart began to beat on day 17. Its rate of beating increased as that of the larval heart decreased, possibly suggesting a gradual shift from a larval heart-driven to a true heart-driven circulation. The true heart was not sensitive to acutely declining P(O(2)) shortly after it began to beat, but increased in activity in response to acutely declining P(O(2)) by day 21. Larval heart responses were similar to those of the true heart, with early insensitivity to declining P(O(2)) (day 13) followed by a response by day 15. Increased velum-driven rotational activity under acutely declining P(O(2)) was greatest in early developmental stages. Together, these findings point to cardiovascular function in L. obtusata larvae being the result of a complex interaction between velum, larval and true heart activities, with the functions of the three structures coinciding but their relative importance changing throughout larval development.

In vivo natriuretic peptide reporter assay identifies chemical modifiers of hypertrophic cardiomyopathy signalling

Despite increased understanding of the fundamental biology regulating cardiomyocyte hypertrophy and heart failure, it has been challenging to find novel chemical or genetic modifiers of these pathways. Traditional cell-based methods do not model the complexity of an intact cardiovascular system and mammalian models are not readily adaptable to chemical or genetic screens. Our objective was to create an in vivo model suitable for chemical and genetic screens for hypertrophy and heart failure modifiers. Using the developing zebrafish, we established that the cardiac natriuretic peptide genes (nppa and nppb), known markers of cardiomyocyte hypertrophy and heart failure, were induced in the embryonic heart by pathological cardiac stimuli. This pathological induction was distinct from the developmental regulation of these genes. We created a luciferase-based transgenic reporter line that accurately modelled the pathological induction patterns of the zebrafish nppb gene. Utilizing this reporter line, we were able to show remarkable conservation of pharmacological responses between the larval zebrafish heart and adult mammalian models. By performing a focused screen of chemical agents, we were able to show a distinct response of a genetic model of hypertrophic cardiomyopathy to the histone deacetylase inhibitor, Trichostatin A, and the mitogen-activated protein kinase kinase 1/2 inhibitor, U0126. We believe this in vivo reporter line will offer a unique approach to the identification of novel chemical or genetic regulators of myocardial hypertrophy and heart failure.

Cardiac remodeling in Drosophila arises from changes in actin gene expression and from a contribution of lymph gland-like cells to the heart musculature

Understanding the basis of normal heart remodeling can provide insight into the plasticity of the cardiac state, and into the potential for treating diseased tissue. In Drosophila, the adult heart arises during metamorphosis from a series of events, that include the remodeling of an existing cardiac tube, the elaboration of new inflow tracts, and the addition of a layer of longitudinal muscle fibers. We have identified genes active in all these three processes, and studied their expression in order to characterize in greater detail normal cardiac remodeling. Using a Transglutaminase-lacZ transgenic line, that is expressed in the inflow tracts of the larval and adult heart, we confirm the existence of five inflow tracts in the adult structure. In addition, expression of the Actin87E actin gene is initiated in the remodeling cardiac tube, but not in the longitudinal fibers, and we have identified an Act87E promoter fragment that recapitulates this switch in expression. We also establish that the longitudinal fibers are multinucleated, characterizing these cells as specialized skeletal muscles. Furthermore, we have defined the origin of the longitudinal fibers, as a subset of lymph gland cells associated with the larval dorsal vessel. These studies underline the myriad contributors to the formation of the adult Drosophila heart, and provide new molecular insights into the development of this complex organ.

Evaluation of the endocrine disrupting potential of the antimicrobial triclosan on early embryonic and larval development in zebrafish.

Triclosan is a widely used chlorinated phenolic antimicrobial used in a variety of soaps, toothpastes, cosmetics, fabrics, and plastics. (Walker and Kimmel 2007). Triclosan is structurally similar to bisphenol A and dioxin, a well documented endocrine disruptor and potent carcinogen, respectively. Triclosan has recently been shown to have endocrine disrupting effects on multiple aquatic organisms and in human cells in culture, including effects on genes regulated by various estrogens, androgens, and thyroid hormones. Current concerns regarding the continued use of triclosan includes its increasing detection in urban streams and its ability to bioaccumulate in the lipid‐dense tissues of exposed organisms. Relevance to the importance of these concerns to humans includes, a report from 2008 demonstrating that 74.6% of human urine samples collected for testing contained triclosan in quantities ranging from 2.3 ug/L to 3, 790 ug/L. Triclosan, considered to be physiologically harmless, may have detrimental effects on organisms during early embryonic development. These effects may vary dramatically based on the timing of exposure. Statistically significant changes in variables such as eye diameter in zebrafish larvae can be observed when exposure times to sublethal concentrations of triclosan (200 ug/L) are prolonged through the first week of embryonic development. Preliminary results also indicate an effect on larval zebrafish heart rates. We hypothesize that this may be the result of an endocrine disrupting effect on the expression of receptors that control heart rate, such as the beta‐adrenergic and muscarinic receptors in the larval heart. Changes in several genes known to be regulated by estrogens, androgens, and thyroid hormones will be analyzed using by RT‐PCR and presented.

<em>Drosophila melanogaster</em> for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval <em>Drosophila melanogaster</em> for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Visualizing the Beating Heart in <em>Drosophila</em>

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Subtle but significant effects of CO2 acidified seawater on embryos of the intertidal snail, Littorina obtusata

Our understanding of the effects of ocean acidification on whole organism function is growing, but most current information is for adult stages of development. Here, we show the effects of reduced pH seawater (pH 7.6) on aspects of the development, physiology and behaviour of encap- sulated embryos of the marine intertidal gastropod Littorina obtusata. We found reduced viability and increased development times under reduced pH conditions, and the embryos had significantly altered behaviours and physiologies. In acidified seawater, embryos spent more time stationary, had slower rotation rates, spent less time crawling, but increased their movement periodicity compared with those maintained under control conditions. Larval and adult heart rates were significantly lower in acidified seawater, and hatchling snails had an altered shell morphology (lateral length and spiral shell length) compared to control snails. Our findings show that ocean acidification may have multi- ple, subtle effects during the early development of marine animals that may have implications for their survival beyond those predicted using later life stages.

Plasticity in the effects of sulfated and nonsulfated sulfakinin on heart contractions

Neuropeptides regulate the frequency of heart contractions. Drosophila melanogaster sulfakinin (drosulfakinin) encodes FDDYGHMRFamide, DSK I, and GGDDQFDDYGHMRFamide, DSK II. Invertebrate sulfakinins are structurally and functionally related to vertebrate cholecystokinins. Naturally-occurring drosulfakinins contain a sulfated or nonsulfated tyrosine and are designated sDSK I, sDSK II, nsDSK I, and nsDSK II. We developed a novel neural-cardiovascular preparation and investigated mechanisms regulating the effect of sulfakinins on D. melanogaster heart. We established the preparation in larva, pupa, and adult to examine plasticity in neural regulation of cardiovascular parameters. We discovered sDSK I increased the frequency of larval, pupal, and adult heart contractions; nsDSK I only increased the frequency of larval contractions, not pupal or adult. We also discovered sDSK II and nsDSK II increased the frequency of larval and adult contractions, not pupal. This is the first report of nonsulfated sulfakinin activity on heart, and sulfakinin activity examined in 3 developmental stages within the same animal species. Our data demonstrate a role for plasticity in the effects of sulfakinins on heart contractions, and suggest multiple mechanisms are involved.

Influence of TCDD on Zebrafish CYP1B1 Transcription during Development

Cytochrome P450 1B1 (CYP1B1) is a heme-containing monooxygenase that metabolizes various polycyclic aromatic hydrocarbons and aryl amines, as well as retinoic acid and steroid hormones. Here we report the cloning of an ortholog of CYP1B1 from zebrafish and the demonstration that transcription of zebrafish CYP1B1 was modulated by two types of mechanisms during different developmental stage. First in late pharyngula stage before hatching, CYP1B1 was constitutively transcribed in retina, midbrain-hindbrain boundary and diencephalon regions through a close coordination between aryl hydrocarbon receptor 2 (AHR2)-dependent and AHR2-independent pathways. After hatching, the basal transcription was attenuated and it could not be elicited upon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. In contrast, TCDD exposure induced de novo CYP1B1 transcription in larval branchial arches and heart tissues via an AHR2-dependent pathway. Blocking AHR2 translation completely eliminated the TCDD-mediated CYP1B1 transcription. However, we did not detect any types of CYP1B1 transcription in liver and kidney tissues through the developmental stage. It suggests that the constitutive and TCDD-inducible types of CYP1B1 transcriptions are modulated by distinct pathways with different tissue specificities. Finally, we investigated the role of CYP1B1 in TCDD-mediated embryonic toxicity. Because knockdown of CYP1B1 did not prevent TCDD-induced pericardial edema and cranial defects, it suggests that CYP1B1 is not involved in the developmental toxicity of dioxin.

Hyped‐up for Hox in Hyderabad

The EMBO Workshop on Upstream and Downstream of Hox Genes took place in Hyderabad, India, between 14 and 17 December 2005, and was organized by R. Mishra and F. Karch. ![][1] For many years, EMBO workshops dealing with new insights into the regulation and function of Hox genes have been held in Switzerland. However, last year, the EMBO World Programme, together with the Centre for Cellular and Molecular Biology (CCMB), held a workshop entitled Upstream and Downstream of Hox Genes in the CCMB's hometown of Hyderabad, India. Imagine beautiful gardens full of butterflies and palm trees, a delightful guest house neighbouring a state‐of‐the‐art research centre, all in a peaceful oasis surrounded by a pulsating and crowded Indian city. The question at the beginning of the meeting was whether the talks and discussions would match the stunning environment; it was answered rapidly and affirmatively. Hox genes have had an illustrious and curious history. Perhaps one of the most famous images in biology from the past century is the four‐winged mutant of the fruit fly Drosophila melanogaster caused by mutations in the Hox gene Ultrabithorax ( Ubx ). This fascinating mutant fly, discovered and characterized by Edward B. Lewis, has motivated many investigators to join the field to understand how these genes are responsible for such complex changes in morphology. Another highlight of Hox history was Matthew Scott's, William McGinnis's and Walter Gehring's discovery of the homeodomain—a 60‐amino‐acid DNA‐binding motif now known to be a key domain in hundreds of genes throughout the animal, fungal and plant kingdoms. Although a lot has been learned through experiments stemming from these important discoveries, there are still many open questions. Among the questions being debated in the field and at the meeting are: How is the regulation of the Hox genes initiated and maintained? How do Hox proteins … [1]: /embed/graphic-1.gif

Direct innervation of the Drosophila melanogaster larval aorta

The heart rate of larval Drosophila is modulated by various biogenic amines and peptides. The actions have always been assumed to be due to direct action on the heart since the larval heart was not known to be innervated. A recent study showed a difference in the sensitivity of the larval heart to serotonin when the CNS was ablated, thus suggesting a direct neural input. Here, we show that GFP tagged motor neurons and nerve terminals are present on the aortic region of the heart. Motor neuron cell bodies also exist outside the CNS. Transmission electron microscopy reveals the direct innervation in the aortic tissue. Thus, developmental and regulatory questions in this genetic model can now be addressed in relation to heart development and neural control.

Cloning and expression of insulin-like growth factors I and II in the shi drum ( Umbrina cirrosa)

Insulin-like growth factors (IGFs) are evolutionarily ancient polypeptides, with potent metabolic actions, affecting cell development and growth. The IGF system consists of two ligands: IGF-I and IGF-II, several binding proteins and high-affinity transmembrane receptors. To understand growth regulation in the teleost shi drum, Umbrina cirrosa, we cloned IGF-I and IGF-II cDNAs, studied their expression and determined the cellular localization of IGF-II peptide by immunohistochemistry. A fragment of 1110 nucleotides, coding for U. cirrosa IGF-I (ucIGF-I), was cloned from liver by PCR. It includes an open reading frame of 561 nucleotides, encoding a 187 amino acid preproIGF-I. A fragment of 938 nucleotides that includes part of the coding sequence and the 3′ UTR of IGF-II (ucIGF-II) was cloned as well. Sequence analysis of ucIGF-I and ucIGF-II showed a high degree of homology with known fish IGF-I and IGF-II. Real-Time PCR showed a higher expression of IGF-I and IGF-II in liver, compared to all other tissues analysed. IGF-II peptide was detected in larval liver, intestine, gills and heart musculature. After metamorphosis, reactivity was particularly evident in the kidney and in red fibres of skeletal muscle. These results add novel information on the nucleotide sequence of IGF-I and IGF-II in a marine teleost, the shi drum, that was recently introduced to the mariculture industry in southern Europe and emphasizes the conservation in the 5′ UTR of IGF-I among teleosts. Furthermore, this study suggests, on the basis of a combined approach of RT-PCR, Real-Time PCR and immunohistochemistry that IGF-I and IGF-II are involved in the regulation of somatic growth in the shi drum.

Direct influence of serotonin on the larval heart of Drosophila melanogaster

The heart rate (HR) of larval Drosophila is established to be modulated by various neuromodulators. Serotonin (5-HT) showed dose-dependent responses in direct application within semi-intact preparations. At 1 nM, HR decreased by 20% while it increased at 10 nM (10%) and 100 nM (30%). The effects plateaued at 100 nM. The action of 5-HT on the heart was examined with an intact Central Nervous System (CNS) and an ablated CNS. The heart and aorta of dorsal vessel pulsate at different rates at rest and during exposure to 5-HT. Splitting the heart and aorta resulted in a dramatic reduction in pulse rate of both the segments and the addition of 5-HT did not produce regional differences. The split aorta and heart showed a high degree of sensitivity to sham changes of saline but no significant effect to 5-HT. Larvae-fed 5-HT (1 mM) did not show any significant change in HR. Since 3,4-methylenedioxymethamphetamine (MDMA) is known to act as a weak agonist on 5-HT receptors in vertebrates, we tested an exogenous application; however, no significant effect was observed to dosage ranging from 1 nM to 100 microM in larvae with and without an intact CNS. In summary, direct application of 5-HT to the larval heart had significant effects in a dose-dependent manner while MDMA had no effect.

Steroid-dependent modification of Hox function drives myocyte reprogramming in theDrosophilaheart

In the Drosophila larval cardiac tube, aorta and heart differentiation are controlled by the Hox genes Ultrabithorax (Ubx) and abdominal A (abdA), respectively. There is evidence that the cardiac tube undergoes extensive morphological and functional changes during metamorphosis to form the adult organ, but both the origin of adult cardiac tube myocytes and the underlying genetic control have not been established. Using in vivo time-lapse analysis, we show that the adult fruit fly cardiac tube is formed during metamorphosis by the reprogramming of differentiated and already functional larval cardiomyocytes, without cell proliferation. We characterise the genetic control of the process, which is cell autonomously ensured by the modulation of Ubx expression and AbdA activity. Larval aorta myocytes are remodelled to differentiate into the functional adult heart, in a process that requires the regulation of Ubx expression. Conversely, the shape, polarity, function and molecular characteristics of the surviving larval contractile heart myocytes are profoundly transformed as these cells are reprogrammed to form the adult terminal chamber. This process is mediated by the regulation of AbdA protein function, which is successively required within these persisting myocytes for the acquisition of both larval and adult differentiated states. Importantly, AbdA specificity is switched at metamorphosis to induce a novel genetic program that leads to differentiation of the terminal chamber. Finally, the steroid hormone ecdysone controls cardiac tube remodelling by impinging on both the regulation of Ubx expression and the modification of AbdA function. Our results shed light on the genetic control of one in vivo occurring remodelling process, which involves a steroid-dependent modification of Hox expression and function.

Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in Drosophila

To analyze the role of cytosolic calcium in regulating heart beat frequency and rhythm, we studied conditional mutations in Drosophila Sarco-endoplasmic reticulum Ca2+-ATPase, believed to be predominantly responsible for sequestering free cytosolic calcium. Abnormalities in the amount or structure of the SERCA protein have been linked to cardiac malfunction in mammals. Drosophila SERCA protein (dSERCA) is highly enriched in Drosophila larval heart with a distinct membrane distribution of SERCA at cardiac Z-lines, suggesting evolutionarily conserved zones for calcium uptake into the sarcoplasmic reticulum. Heart beat frequency is strikingly reduced in mutant animals following dSERCA inactivation, (achieved by a brief exposure of these conditional mutants to non-permissive temperature). Cardiac contractions also show abnormal rhythmicity and electrophysiological recordings from the heart muscle reveal dramatic alterations in electrical activity. Overall, these studies underscore the utility of the Drosophila heart to model SERCA dysfunction dependent cardiac disorders and constitute an initial step towards developing Drosophila as a viable genetic model system to study conserved molecular determinants of cardiac physiology.

Heartbeat patterns during the postembryonic development of Drosophila melanogaster

Pulsations of the dorsal vessel were recorded in vivo during the whole postembryonic development of D. melanogaster, by means of a newly invented, pulse-light opto-cardiographic method. The young larvae of the 1st and 2nd instars submerged in the feeding medium exhibited extremely high rates of heartbeat, 7 Hz at room temperature. These values are among the highest rates of heartbeat ever recorded in the animal kingdom. The fully grown larvae of the 3rd instar showed approximately half of the maximum heartbeat rate (3.5–4 Hz), which became stabilized after pupariation to 2.5–2.7 Hz.The larval heartbeat was always uni-directional, in the forward-oriented or anterograde direction and it was almost continuous. The slowly disintegrating, old larval heart used to beat at the constant frequency of 2.5–2.7 Hz until complete cessation of all cardiac functions in 1-day-old puparium. In spite of the persisting constant heartbeat frequency, the transformation process of the larval heart was associated with successively decreasing amplitude of the systolic contractions and with the prolongation of the resting periods. The newly formed heart of the pupal–adult structure exhibited a qualitatively new pattern of heartbeat activity, which was manifested by periodic reversal of the heartbeat with the faster anterograde and slower retrograde phases. The frequencies of both of these reciprocal cardiac pulsations gradually increased during the advanced pharate adult period, reaching the values of 4–5 Hz at the time of adult eclosion. Adult males and females also exhibited a perfect pattern of heartbeat reversal, with still very high rates of the anterograde heartbeat, in the range of 5–6 Hz. In addition to the cardiac functions, we have recorded several kinds of extracardiac pulsations, which often interfered severely with the recordings of the heartbeat. There were strong, irregular extracardiac pulsations of a neurogenic nature (somatic muscles, oral armature) and relatively slow extracardiac pulsations of a myogenic nature (intestinal peristaltics, 0.2–0.3 Hz). The extracardiac and cardiac pulsations were independent, their functions were not correlated. A possibility of creating new challenges in combination of molecular biology with the functional physiology of the heart have been discussed.

A nonpeptide provides insight into mechanisms that regulate Drosophila melanogaster heart contractions

Here we report the effect of a nonpeptide, benzethonium chloride (bztc), on Drosophila melanogaster larval, pupal, and adult heart rates in vivo. Benzethonium chloride reduced the frequency of spontaneous contractions in the D. melanogaster pupal heart, but not in the larval heart or the adult heart as measured in noninvasive whole animal preparations. When applied directly to the D. melanogaster heart, in the absence of hemolymph, bztc reduced the frequency of spontaneous contractions in larval, pupal, and adult hearts. These findings are consistent with the conclusion that bztc acts through or is regulated by different mechanisms in these three developmental stages. An alternative explanation is that larval hemolymph and adult hemolymph contain a material that interferes with the effect of the nonpeptide on heart contractions. Bztc mimicked the effect of the peptide dromyosuppressin (DMS) on the heart at an equivalent concentration; in contrast, 103-fold more nonpeptide is required to mimic the effect of DMS on fly gut. These findings are consistent with the presence of tissue-specific myosuppressin receptors or mechanisms.