Daniela Amelio

The structural characteristics of the heart ventricle of the African lungfish Protopterus dolloi: freshwater and aestivation

This paper reports on the structure and ultrastructure of the ventricular myocardium of the African lungfish Protopterus dolloi in freshwater (FW), in aestivation (AE), and after the AE period. The myocardium shows a conventional myofibrillar structure. All the myocytes contain large intracytoplasmic spaces occupied by a pale material that could contain glycosaminoglycans and/or glycogen, which may be used as food and water reservoirs. In FW, the myocytes in the trabeculae associated with the free ventricular wall show structural signs of low transcriptional and metabolic activity (heterochromatin, mitochondria of the dense type). These signs are partially reversed during the AE period (euchromatin, mitochondria with a light matrix), with a return to the FW appearance after arousal. The myocytes in the septum show, in FW conditions, nuclear polymorphism (heterochromatin, euchromatin), and two types (colliquative and coagulative) of necrosis. In AE, all the septal myocytes show euchromatin, and the number of necrotic cells increases greatly. Cell necrosis appears to be related to the septal architecture. After arousal, the septal myocytes exhibit a heterochromatin pattern, the number of necrotic cells decreases, cell debris accumulates under the endocardium, and phagocytosis takes place. Despite being a morphologic continuum, the trabeculae associated with the free ventricular wall appear to constitute a different compartment from that formed by the trabeculae in the ventricular septum. Paradoxically, AE appears to trigger an increase in transcriptional and synthetic myocardial activities, especially at the level of the ventricular septum. This activity may be involved in mechanisms of autocrine/paracrine regulation. Aestivation cannot be regarded as the result of a general depression of all cellular and organic activities. Rather, it is a much more complex state in which the interplay between upregulation and downregulation of diverse cell activities appears to play a fundamental role.

Catestatin increases the expression of anti-apoptotic and pro-angiogenetic factors in the post-ischemic hypertrophied heart of SHR.

Background In the presence of comorbidities the effectiveness of many cardioprotective strategies is blunted. The goal of this study was to assess in a hypertensive rat model if the early reperfusion with anti-hypertensive and pro-angiogenic Chromogranin A-derived peptide, Catestatin (CST:hCgA352–372; CST-Post), protects the heart via Reperfusion-Injury-Salvage-Kinases (RISK)-pathway activation, limiting infarct-size and apoptosis, and promoting angiogenetic factors (e.g., hypoxia inducible factor, HIF-1α, and endothelial nitric oxide synthase, eNOS, expression). Methods and Results The effects of CST-Post on infarct-size, apoptosis and pro-angiogenetic factors were studied in isolated hearts of spontaneously hypertensive rats (SHR), which underwent the following protocols: (a) 30-min ischemia and 120-min reperfusion (I/R); (b) 30-min ischemia and 20-min reperfusion (I/R-short), both with and without CST-Post (75 nM for 20-min at the beginning of reperfusion). In unprotected Wistar-Kyoto hearts, used as normal counterpart, infarct-size resulted smaller than in SHR. CST-Post reduced significantly infarct-size and improved post-ischemic cardiac function in both strains. After 20-min reperfusion, CST-Post induced S-nitrosylation of calcium channels and phosphorylation of RISK-pathway in WKY and SHR hearts. Yet specific inhibitors of the RISK pathway blocked the CST-Post protective effects against infarct in the 120-min reperfusion groups. Moreover, apoptosis (evaluated by TUNEL, ARC and cleaved caspase) was reduced by CST-Post. Importantly, CST-Post increased expression of pro-angiogenetic factors (i.e., HIF-1α and eNOS expression) after two-hour reperfusion. Conclusions CST-Post limits reperfusion damages and reverses the hypertension-induced increase of I/R susceptibility. Moreover, CST-Post triggers antiapoptotic and pro-angiogenetic factors suggesting that CST-Post can be used as an anti-maladaptive remodeling treatment.

Morphological and physiological study of the cardiac NOS/NO system in the Antarctic (Hb−/Mb−) icefish Chaenocephalus aceratus and in the red-blooded Trematomus bernacchii

The nitric oxide synthase (NOS)/nitric oxide (NO) system integrates cellular biochemical machinery and energetics. In heart microenvironment, dynamic NO behaviour depends upon the presence of superoxide anions, haemoglobin (Hb), and myoglobin (Mb), being hemoproteins are major players disarming NO bioactivity. The Antarctic icefish, which lack Hb and, in some species, also cardiac Mb, represent a unique model for exploring Hb and Mb impact on NOS/NO function. We report in the (Hb(-)/Mb(-)) icefish Chaenocephalus aceratus the presence of cardiac NOSs activity (NADPH-diaphorase) and endothelial NOS (eNOS)/inducible NOS (iNOS) zonal immuno-localization in the myocardium. eNOS is localized on endocardium and, to a lesser extent, in myocardiocytes, while iNOS is localized exclusively in myocardiocytes. Confronting eNOS and iNOS expression in Trematomus bernacchii (Hb(+)/Mb(+)), C. hamatus (Hb(-)/Mb(+)) and C. aceratus (Hb(-)/Mb(-)) is evident a lower expression in the Mb-less icefish. NO signaling was analyzed using isolated working heart preparations. In T. bernacchii, L-arginine and exogenous (SIN-1) NO donor dose-dependently decreased stroke volume, indicating decreased inotropism. L-arginine-induced inotropism was NOSs-dependent, being abolished by NOSs-inhibitor NG-monomethyl-L-arginine (L-NMMA). A SIN-1-induced negative inotropism was found in presence of SOD. NOS inhibition by L-N5-N-iminoethyl-L-ornithine (L-NIO) and L-NMMA confirmed the NO-mediated negative inotropic influence on cardiac performance. In contrast, in C. aceratus, L-arginine elicited a positive inotropism. SIN-1 induced a negative inotropism, which disappeared in presence of SOD, indicating peroxynitrite involvement. Cardiac performance was unaffected by L-NIO and L-NIL. NO signaling acted via a cGMP-independent mechanism. This high conservation degree of NOS localization pattern and signaling highlights its importance for cardiac biology.

The conus valves of the adult gilthead seabream (Sparus auratus)

The conus (bulbo‐ventricular) valves of teleosts perform a key function in the control of blood backflow during ventricular diastole. However, the structural characteristics of these valves are almost unknown. This paper presents a systematic anatomical, histological and structural study of the conus valves of the adult gilthead seabream (Sparus auratus). S. auratus shows two major left and right valves consisting of the leaflet and the supporting sinus. Each valvar leaflet can be divided into a stout proximal body and a flap‐like distal region. The proximal body is structured into three layers: a luminal fibrosa, a dense cellular core and a parietal fibrosa. The luminal fibrosa is a collagenous structure extending the entire length of the leaflet, while the parietal fibrosa is restricted to the most proximal area. The dense cellular core consists of fibroblastic cells and a matrix rich in glycoconjugates, collagen and elastin. The histochemical and structural data suggest that the luminal fibrosa bears most of the force associated with valvar closure, while the cellular core acts as a cushion dampening vibrations and absorbing the elastic recoil. The sinus wall is a fibrous layer which shows proximal–distal differences in thickness. It also shows compositional differences that can be related to mechanical function. We describe the presence of a fibrous cylinder formed by the sinus wall, the fibrous interleaflet triangles and the fibrous layer that covers the inner surface of the conus myocardium. This fibrous cylinder constitutes the structural nexus between the ventricle, the conus and the bulbus arteriosus, provides support for the conus valves and separates the valvar complex from the surrounding tissues. The structure of the conus valves in S. auratus is different from that found in other vertebrates. Anatomical similarities between the conus valves and the mammalian arterial valves are emphasized. Each phyletic group appears to have developed specific structures in order to perform similar functions.

Nitric oxide synthase-dependent “On/Off” switch and apoptosis in freshwater and aestivating lungfish, Protopterus annectens: Skeletal muscle versus cardiac muscle

African lungfishes (Protopterus spp.) are obligate air breathers which enter in a prolonged torpor (aestivation) in association with metabolic depression, and biochemical and morpho-functional readjustments during the dry season. During aestivation, the lungfish heart continues to pump, while the skeletal muscle stops to function but can immediately contract during arousal. Currently, nothing is known regarding the orchestration of the multilevel rearrangements occurring in myotomal and myocardial muscles during aestivation and arousal. Because of its universal role in cardio-circulatory and muscle homeostasis, nitric oxide (NO) could be involved in coordinating these stress-induced adaptations. Western blotting and immunofluorescence microscopy on cardiac and skeletal muscles of Protopterus annectens (freshwater, 6months of aestivation and 6days after arousal) showed that expression, localization and activity of the endothelial-like nitric oxide synthase (eNOS) isoform and its partners Akt and Hsp-90 are tissue-specifically modulated. During aestivation, phospho-eNOS/eNOS and phospho-Akt/Akt ratios increased in the heart but decreased in the skeletal muscle. By contrast, Hsp-90 increased in both muscle types during aestivation. TUNEL assay revealed that increased apoptosis occurred in the skeletal muscle of aestivating lungfish, but the myocardial apoptotic rate of the aestivating lungfish remained unchanged as compared with the freshwater control. Consistent with the preserved cardiac activity during aestivation, the expression of apoptosis repressor (ARC) also remained unchanged in the heart of aestivating and aroused fish as compared with the freshwater control. Contrarily, ARC expression was strongly reduced in the skeletal muscle of aestivating lungfish. On the whole, our data indicate that changes in the eNOS/NO system and cell turnover are implicated in the morpho-functional readjustments occurring in lungfish cardiac and skeletal muscle during the switch from freshwater to aestivation, and between the maintenance and arousal phases of aestivation.

HSP90 and eNOS partially co‐localize and change cellular localization in relation to different ECM components in 2D and 3D cultures of adult rat cardiomyocytes

Background information. Cultivation techniques promoting three‐dimensional organization of mammalian cells are of increasing interest, since they confer key functionalities of the native ECM (extracellular matrix) with a power for regenerative medicine applications. Since ECM compliance influences a number of cell functions, Matrigel‐based gels have become attractive tools, because of the ease with which their mechanical properties can be controlled. In the present study, we took advantage of the chemical and mechanical tunability of commonly used cell culture substrates, and co‐cultures to evaluate, on both two‐ and three‐dimensional cultivated adult rat cardiomyocytes, the impact of ECM chemistry and mechanics on the cellular localization of two interacting signalling proteins: HSP90 (heat‐shock protein of 90 kDa) and eNOS (endothelial nitric oxide synthase).

Effects of temperature on the nitric oxide-dependent modulation of the Frank-Starling mechanism: the fish heart as a case study.

The Frank-Starling law is a fundamental property of the vertebrate myocardium which allows, when the end-diastolic volume increases, that the consequent stretch of the myocardial fibers generates a more forceful contraction. It has been shown that in the eel (Anguilla anguilla) heart, nitric oxide (NO) exerts a direct myocardial relaxant effect, increasing the sensitivity of the Frank-Starling response (Garofalo et al., 2009). With the use of isolated working heart preparations, this study investigated the relationship between NO modulation of Frank-Starling response and temperature challenges in the eel. The results showed that while, in long-term acclimated fish (spring animals perfused at 20 °C and winter animals perfused at 10 °C) the inhibition of NO production by L-N5 (1-iminoethyl)ornithine (L-NIO) significantly reduced the Frank-Starling response, under thermal shock conditions (spring animals perfused at 10 or 15 °C and winter animals perfused at 15 or 20 °C) L-NIO treatment resulted without effect. Western blotting analysis revealed a decrease of peNOS and pAkt expressions in samples subjected to thermal shock. Moreover, an increase in Hsp90 protein levels was observed under heat thermal stress. Together, these data suggest that the NO synthase/NO-dependent modulation of the Frank-Starling mechanism in fish is sensitive to thermal stress.

Signal molecule changes in the gills and lungs of the African lungfish Protopterus annectens, during the maintenance and arousal phases of aestivation

African lungfishes are obligate air breathers, with reduced gills and pulmonary breathing throughout their life. During the dry season they aestivate on land, with the collapse of secondary lamellae of their gills and the establishment of an exclusive aerial ventilation through the vascularization and expansion of their lungs. To date, the mechanisms underlining the respiratory organ remodeling in aestivating lungfishes are unknown. This study aimed to identify key switch components of the stress-induced signal transduction networks implicated in both rapid and medium-long term remodeling of the gills and lungs of the African lungfish Protopterus annectens during aestivation. Through immunofluorescence microscopy and Western blotting, the localization and the expression of nitric oxide synthase (NOS), Akt, Hsp-90 and HIF-1α were evaluated in both gills and lungs exposed to three experimental conditions: freshwater (FW), 6 months of experimentally induced aestivation (6mAe), and 6 days after arousal from 6 months of aestivation (6mAe6d). After 6mAe, the expression of NOS (p-eNOS antibody), Akt (p-Akt antibody), and Hsp-90 decreased in the gills, while NOS and Hsp-90 expression increased with Akt remained unchanged in the lungs. Upon 6mAe6d, NOS, Akt and Hsp-90 expression in the gills returned to the respective FW values. In the lungs of the aroused fish, NOS and Akt decreased to their respective FW levels, while Hsp-90 expression was enhanced with respect to aestivation. In both respiratory organs, the qualitative and quantitative patterns of HIF-1α expression correlated inversely to those of NOS. Overall, our findings suggest that the molecular components of the NOS/NO system changed in a tissue-specific manner in parallel with organ readjustment in the gills and lungs of P. annectens during aestivation and arousal.

Protective role of GPER agonist g-1 on cardiotoxicity induced by doxorubicin.

The use of Doxorubicin (Dox), a frontline drug for many cancers, is often complicated by dose‐limiting cardiotoxicity in approximately 20% of patients. The G‐protein estrogen receptor GPER/GPR30 mediates estrogen action as the cardioprotection under certain stressful conditions. For instance, GPER activation by the selective agonist G‐1 reduced myocardial inflammation, improved immunosuppression, triggered pro‐survival signaling cascades, improved myocardial mechanical performance, and reduced infarct size after ischemia/reperfusion (I/R) injury. Hence, we evaluated whether ligand‐activated GPER may exert cardioprotection in male rats chronically treated with Dox. 1 week of G‐1 (50 μg/kg/day) intraperitoneal administration mitigated Dox (3 mg/kg/day) adverse effects, as revealed by reduced TNF‐α, IL‐1β, LDH, and ROS levels. Western blotting analysis of cardiac homogenates indicated that G‐1 prevents the increase in p‐c‐jun, BAX, CTGF, iNOS, and COX2 expression induced by Dox. Moreover, the activation of GPER rescued the inhibitory action elicited by Dox on the expression of BCL2, pERK, and pAKT. TUNEL assay indicated that GPER activation may also attenuate the cardiomyocyte apoptosis upon Dox exposure. Using ex vivo Langendorff perfused heart technique, we also found an increased systolic recovery and a reduction of both infarct size and LDH levels in rats treated with G‐1 in combination with Dox respect to animals treated with Dox alone. Accordingly, the beneficial effects induced by G‐1 were abrogated in the presence of the GPER selective antagonist G15. These data suggest that GPER activation mitigates Dox‐induced cardiotoxicity, thus proposing GPER as a novel pharmacological target to limit the detrimental cardiac effects of Dox treatment. J. Cell. Physiol. 232: 1640–1649, 2017.

Nitric oxide modulates the frog heart ventricle morphodynamics

The aim of this work was to investigate in the avascular heart of the frog Rana esculenta the influence of nitric oxide (NO) on ventricular systolic and diastolic functions by using a novel image analysis technique. The external volume variations of the whole ventricle were monitored during the heart cycle by video acquisition(visible light) and analysed by an appropriately developed software with a specific formula for irregular convex solids. The system, which measures the rate of volume changes and the ejection fraction, directly determined the volumetric behaviour of the working frog heart after stimulation or inhibition of NOS-NOcGMP pathway. End-diastolic volume (EDVext), end-systolic volume (ESVext), contraction and relaxation velocities (dV/dtsys and dV/dtdia, respectively), stroke volume (SV) and ejection fraction (EF), were measured before and after perfusion with NOS substrate (L-arginine), NO donor (SIN-1), cGMP analogue (8-Br-cGMP),NOS inhibitors (NG-monomethyl-L-arginine, L-NMMA; L-N(5)-(1-iminoethyl)-ornithine, L-NIO; 7-Nitroindazole,7-NI) and guanylyl cyclase inhibitor (ODQ). The results showed that NO reduces ventricular systolicfunction improving diastolic filling, while NOS inhibition increases contractility impairing ventricular filling capacity. The presence of activated eNOS (p-eNOS) was morphologically documented, further supporting that the mechanical activity of the ventricular pump in frog is influenced by a tonic release of NOS-generated NO.