Effects of feeding and digestion on myocardial contractility and expression of calcium-handling proteins in Burmese pythons (Python molurus)

Comparative Biochemistry and Physiology, Part B 240 (2020) 110371

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Effects of feeding and digestion on myocardial contractility and expression of calcium-handling proteins in Burmese pythons (Python molurus)

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Eliton da Silva Vasconcelosa, Ana Lúcia Kalinina, Rafael Correa Ciprianoa, Samuel dos Santos Beserrab, André Guelli Lopesa, Cléo Alcântara da Costa Leitea, ⁎ Diana Amaral Monteiroa, a b

Department of Physiological Sciences, Federal University of São Carlos (UFSCar), São Carlos, São Paulo, Brazil Departament of Biophysics, Federal University of São Paulo - UNIFESP, São Paulo, SP, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Specific dynamic action Cardiac function Snake SERCA NCX PLB

Pythons are important models of studies on postprandial metabolism because their physiological responses are exacerbated when digesting large prey. Prior studies of these animals have shown hypertrophy of the cardiac tissue 2 to 3 days after feeding, coinciding with the peak of the specific dynamic action (SDA), but the consequences of this remodeling in myocardial contractility have not been studied, which is the purpose of this work. Specimens of Python molurus were divided into two groups: a Digesting group (2 days after feeding, at the peak of SDA), and a Fasting group (28 days after feeding). When compared to the Fasting group, the Digesting group showed higher relative ventricular mass and calcium-handling protein expression such as sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), and the Na+/Ca2+ exchanger (NCX). Digesting pythons also exhibited significant increases in the cardiac contraction force (Fc), rates of force development and relaxation, and cardiac pumping capacity. Therefore, the higher SERCA, PLB and NCX expression levels increased cytosolic Ca2+ transient amplitude, improving myofilament force. These changes are crucial to maintain cardiac output and a relatively high and continuous blood flow required by metabolic expenditure that occurs in postprandial animals.

1. Introduction Squamate reptiles have the ability to ingest large prey, notably some species of snakes can ingest prey equivalent to 100% of their body weight (Secor and Diamond, 1997). In such species, this capacity is associated with the ability to survive long periods of food deprivation, making these animals an interesting model to study specific dynamic action (SDA), the increase in the metabolic rate associated with digestion, assimilation and biosynthesis (Brody, 1945; Kleiber, 1975; Jobling, 1981). The postprandial increase in metabolic rate is assumed generally up to 50% in humans, 320% in most other animals and up to 1600% in foraging snakes with the “sit and wait” behavior, i.e., the predator waits until the prey comes close (Brody, 1945; Jobling, 1981; Hailey and Davies, 1987; Westerterp-Plantenga et al., 1992; Kalarani and Davies, 1994; Janes and Chappell, 1995; Secor and Diamond, 1995). Experiments conducted with Python molurus showed increases of about 40 fold in the resting metabolic rate after feeding the equivalent of 100% of

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their body weight. Furthermore, respiratory metabolism remains high for about 15 days (Secor and Diamond, 1997). Thus, the large increase in the metabolic rate causes significant increases in oxygen consumption. In the case of ingestion of large prey, this increase can persist for several days, requiring major adjustments in both uptake and transport of oxygen in order to prevent, for example, an overload in the cardiovascular system. Regarding high metabolic activity during digestion, the heart must remain intact to some extent in its contractile function, especially in order to maintain an increasing demand related to increased metabolic activity. This would be essential during refeeding, when the metabolic rate rises after prey ingestion, even before the nutrients are digested and absorbed. During the SDA, digesting large prey requires a higher metabolic increase than that caused by forced exercise and this lasts for several days (Secor and Diamond, 1995, 1997). Thus, investigations on the functional aspects of cardiac muscle contractility in snakes, animals that can be studied in terms of extreme metabolic conditions, are of particular interest. Andersen et al. (2005) investigated the nature of cardiac

Corresponding author at: Department of Physiological Science, Federal University of São Carlos, Via Washington Luis, Km 235, 13566-905 São Carlos, SP, Brazil. E-mail address: [email protected] (D.A. Monteiro).

https://doi.org/10.1016/j.cbpb.2019.110371 Received 26 June 2019; Received in revised form 7 October 2019; Accepted 14 October 2019 Available online 30 October 2019 1096-4959/ © 2019 Elsevier Inc. All rights reserved.

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2. Material and methods

analyses were performed in duplicate. Proteins were quantified using the Bradford Protein Assay (Bio-Rad, CA, USA). Samples of 100 μg of protein were subjected to SDS-PAGE 8% (NCX and SERCA) or 12% (PLB) and then transferred to a PVDF membrane overnight at 4 °C, using a Mini Trans-Blot Transfer Cell system (Bio-Rad, Laboratories, USA) containing Tris 25 mM, glycine 190 mM, methanol 20% and SDS 0.05%. Subsequently, membranes were blocked with 5% non-fat dry milk in Tris-buffered saline plus 0.1% Tween-20 for 60 min at room temperature. Then, membranes were incubated overnight at 4 °C with anti-NCX1 rabbit polyclonal antibodies (0.5 μg ml−1, sc32881, Santa Cruz Biotechnology Inc., CA, USA), anti-SERCA2 goat polyclonal antibodies (0.5 μg ml−1, sc-8095, Santa Cruz Biotechnology Inc., CA, USA), and anti-PLB (phosphorylated and non-phosphorylated phospholamban) mouse monoclonal antibodies (0.15 μg ml−1, 05-205, Millipore Corporation, Billerica, MA). After washing (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20), the membranes were incubated for 90 min at room temperature with the secondary antibody alkaline phosphatase-conjugated: goat anti-rabbit IgG (0.2 μg ml−1, sc-2034, Santa Cruz Biotechnology Inc., CA, USA) or goat anti-mouse IgG (0.34 μg ml−1, AP308A, Upstate Chemicon, USA). Membranes were washed and the immunocomplexes were detected using the alkaline phosphatase conjugate substrate kit (Bio-Rad, CA, USA). Integrated optical densities were measured with ImageJ software from the scanned membranes (Abramoff et al., 2004). The results were expressed in arbitrary units (A.U.) of optical density normalized by actin I-19 (0.5 μg ml−1, sc-1616, Santa Cruz Biotechnology Inc., CA, USA).

2.1. Animals

2.4. Cardiac contractility ex vivo

Specimens of Python molurus (Wt = 1345.2 ± 151.9 g) were provided by the Jacarezário Laboratory, Department of Zoology, São Paulo State University (UNESP), Rio Claro, SP, Brazil. For at least 30 days prior to experimentation, the snakes were maintained in plastic cages within a temperature-controlled room at 30 °C ± 2 °C, under natural photoperiod, with free access to water and fed on rats every other week. After this acclimation period, the animals were divided into two groups with different feeding regimes: Fasting: specimens in post-absorptive period (28 days after feeding; n = 10), and digesting: specimens in postprandial period, i.e., in the peak of SDA (two days after consuming rats equal to 30% body mass; n = 10). This food ratio was based on the studies of Secor and Diamond (1998), Starck and Beese (2001), and Andersen et al. (2005).

The pythons were sacrificed and the hearts were surgically removed and immediately transferred to an ice-cold physiological solution. Strips with a maximal thickness of 4 mm were excised from the ventricle, tied at each end to two platinum hooks, and placed into an oxygenated bathing medium containing (mM): 100 NaCl, 5 KCl, 1.2 MgSO4, 1.5 NaH2PO4, 27 NaHCO3, 2.5 CaCl2 and 10 glucose. The solution was continuously bubbled with a gas mixture of 2% CO2 and 98% O2 providing a pH of 7.5 during recordings. This physiological solution was kept at 30 °C by a temperature-controlled water bath. One platinum hook was attached to a LETICA isometric force transducer (Letica Corporation, USA) through a stainless-steel wire and the other end tied around a platinum electrode connected to an AVS 100D stimulator (Solução Integrada Ltda., São Paulo, Brazil) which delivered electrical pulses (duration of 8 ms and a voltage 50% above the threshold) assuring maximal stimulation throughout the experiment. Preparations were stretched to obtain a twitch tension at the maximum of the lengthtwitch tension relation (Kalinin and Gesser, 2002; Rocha et al., 2007). Isometric contractions from the force transducers were recorded by a data-acquisition system (Soft & Solutions - Solução Integrada Ltda., São Paulo, Brazil). The length and wet mass of each strip were measured and the isometric force (Fc) relative to the cross-sectional area (mN mm−2) was calculated assuming a muscle density of 1.06 mg mm−3 (Layland et al., 1995). The rates of tension development and relaxation (maximum derivative from the recorded force-time curve during the contraction phase (+dF/dt - mN ms−1) and during the relaxation phase (−dF/dt – mN ms−1), respectively) were also evaluated. The twitch tension was then stabilized for about 40 min at 0.2 Hz (12 bpm). After the stabilization period, ventricle strip preparations were subjected to increases in the imposed contraction frequency from 0.2 Hz (12 bpm) until the frequency in which at least 80% of the strips were still able to contract regularly. To investigate the role of the sarcoplasmic reticulum (SR) on cardiac contractility, the protocols described above were repeated in the presence of 10 μmol l−1 ryanodine (Ry). To obtain a more integrative perspective on the effects of SDA in heart function, the cardiac pumping capacity (CPC) at each stimulation

hypertrophy in ventricles from three groups of Burmese pythons: fasting (fast of 28 days), digesting (two days after consuming rats equal to ~25% body mass) and postdigestion (28 days after the meal). They recorded sevenfold increases in oxygen consumption and 40% increases in ventricular mass during digestion. These authors sequenced the isoforms of cardiac myosin heavy chains and found a significant increase in the expression of messenger RNA for heavy-chain cardiac myosin during digestion. They concluded that the newly synthesized protein results from increased transcription of the gene encoding cardiac myosin heavy chains and that cardiac hypertrophy follows from de novo addition of contractile elements. These authors suggested that this cardiac hypertrophy possibly has important consequences for oxygen transport and could explain why the stroke volume measured by Secor et al. (2000) in postprandial pythons is greater than that measured in fasted animals doing maximal exercise. Sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB) and the Na+/Ca2+ exchanger (NCX) are key Ca2+ cycling proteins in the process of cardiac excitation-contraction coupling (ECC). The ubiquitous second messenger Ca2+ is essential in cardiac electrical activity and is the direct activator of myofilaments, which cause contraction (Bers, 2002; Bers, 2001). The postprandial morphological and physiological changes in different organs have been described in pythons, but the effects of cardiac remodeling on myocardial contractility and expression of Ca2+ cycling proteins remains unclear, which motivated the present study.

2.2. Relative ventricular mass (RVM) Animals of both experimental groups were sacrificed, and the body mass was measured (Wt - g). Ventricles were carefully excised and weighed (Wv - g) to obtain the ventricular mass (relative ventricular mass, RVM - % of Wt), and immediately transferred to an ice-cold physiological solution. Subsequently, ventricles were divided for the two experimental proposes: ex vivo measurements of force contraction and protein expression. For protein expression, parts of each ventricle were immediately frozen in liquid nitrogen and posteriorly stored at −80 °C. 2.3. Cardiac protein expression (SERCA, PLB and NCX) Western blotting was performed as described by Bocalini et al. (2012) and Monteiro et al. (2016). Frozen ventricles were homogenized in hyperosmotic buffer (250 mM sucrose, 50 mM Tris, 1 mM EDTA pH 7.4), using an IKA homogenizer (T10 basic ULTRA-TURRAX®) and centrifuged at 10,000g for 40 min at 4 °C. In order to obtain the microsomal fraction, the supernatants were ultracentrifuged (100,000g, 1 h) and the membrane pellet was resuspended in a small volume (100 to 150 μl) of Tris-EDTA buffer (50 mM Tris, 1 mM EDTA, pH 7.4). All 2

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The Fasting group did not exhibit differences in the Fc values after treatment with ryanodine in all the tested stimulation frequencies (Fig. 4A). On the other hand, a leftward and downward shift in the force-frequency relationship (FFR) was detected after treatment with ryanodine in the Digesting groups (Fig. 4B). The proportion of the SR Ca2+ contribution to ECC in the myocardium of the Digesting group was approximately 42% from 0.2 to 0.4 Hz. Above these frequencies, the SR contributed 16.3% to tension generation. When compared to the Fasting group, the relative participation of SR in total force-associated Ca2+ flux following feeding was marked higher (ranging from 40 to 800%) at low stimulation frequencies (0.2 to 0.6 Hz). After treatment with Ry, the Digestion group displayed significant reductions of +dF/dT (~50% between 0.2 and 1.0 Hz,) and −dF/dT (~46% between 0.2 and 0.6 Hz) (Fig. 5B), while the Fasting group did not show significant differences in these parameters, except for significantly lower +dF/dT values below 0.4 Hz (Fig. 5A). The maximum contraction and relaxation derivatives from Fasting Ry and Digesting Ry groups remained constant throughout the increments in the stimulation frequency, when compared to the initial values (0.2 Hz).

frequency was calculated as described by Matikainen and Vornanen (1992). 2.5. Statistical analysis Results are presented as means ± S.E.M. All data were assessed for normality with a Shapiro-Wilk normality test prior to additional statistical analysis. Differences in protein expressions levels and relative ventricular mass between the Fasting and Digesting groups were identified using non-paired t-test or Mann-Whitney test were used (GraphPad Instat 3.1, GraphPad Software, Inc.). To compare changes in contractility parameters over time or during increments of stimulation frequency, a two-way repeated-measures ANOVA with Holm-Sidak's test for multiple comparisons was performed (Sigma Stat 3.5, Systat Software, Inc.). Differences between means at a 5% level (P < 0.05) were considered significant. 3. Results The relative ventricular mass (RVM) of the Digesting group was significantly higher (0.145 ± 0.004%) than the Fasting group ones (0.118 ± 0.005%). Ca2+ handling proteins (SERCA2a, PLB, and NCX1) were investigated in the ventricular myocardium (Fig. 1). Both experimental groups expressed the NCX1 (Fig. 1A) with 37 kDa and PLB with 25 kDa (Fig. 1B). On the other hand, the SERCA2a protein was expressed with 100 kDa by the Digesting group and with 105 kDa by the Fasting group (Fig. 1C). The Digesting group showed increased SERCA2a (326%), PLB (158%) and NCX1 (145%) protein expression when compared to the Fasting group. Furthermore, in the ventricle of postprandial pythons, the SERCA2a/PLB ratio was 2.0 times higher than those values from the Fasting group. Fig. 2A shows that the experimental time of 40 min did not change the isometric force (Fc) development of ventricular strips from both experimental groups. However, the Fc values recorded for the Digesting group (17.0 ± 1.7 mN mm−2) were significantly higher (~100%) than the Fasting ones (8.5 ± 0.7 mN mm−2). The rates of contraction (+dF/dt) and relaxation (−dF/dT) over time are shown in Fig. 1B. Both +dF/dT and −dF/dT were maintained constant along the experimental time, and were significantly higher (~75% and 40%, respectively) in the Digesting group. The effects of increasing stimulation frequency on force development of the Fasting and Digesting groups are shown in Fig. 3A. The Fasting group maintained a constant Fc from 0.2 to 0.4 Hz (9.2 ± 0.7 mN mm−2), above which it decreased significantly, reaching minimum values at the highest sustained frequency of 1.4 Hz (3.8 ± 0.4 mN mm−2). In the Digesting group, the Fc was also maintained constant (18.1 ± 1.2 mN mm−2) from 0.2 to 0.4 Hz, decreasing significantly and progressively with subsequent increases in stimulation frequency, reaching minimum values (5.3 ± 0.6 mN mm−2) at the highest sustained frequency of 1.6 Hz. The Fc values of the Digesting group were significantly higher (~88%) than the Fasting ones in all tested frequencies. The effects of increments in the stimulation frequency on the rates of contraction and relaxation are shown in Fig. 3B. The Fasting group did not exhibit differences in the rates of contraction and relaxation in all stimulation frequencies. The Digesting group maintained a constant −dF/dT in all frequencies, but +dF/dT values decreased significantly above 1.2 Hz. When compared to the Fasting group, the Digesting group presented significantly higher values of +dF/dT (83% between 0.2 and 1.2 Hz) and −dF/dT (38% between 0.2 and 1.0 Hz). Moreover, the Digesting group exhibited significantly higher CPC values (~76%) in stimulation frequencies between 0.8 and 1.2 Hz (Fig. 3C). The relative contribution of sarcoplasmic reticulum (SR) to total force development during increments in the stimulation frequency is presented in Fig. 4.

4. Discussion In this report, we describe an improvement in the contractile function with concomitant increases in the relative ventricle mass and Ca2+-handling protein expression levels in fed Burmese python heart (48 h after feeding, in the peak of SDA). According to Secor (2009), SDA is an integral part of an organism's energy budget caused from a series of integrative physiological processes that results in the digestion, absorption, and assimilation of a meal. During fasting, pythons downregulated metabolic and physiological functions showing atrophy of organs (such as the heart, kidney, and liver), which is massively upregulated to accommodate the digestion of prey (Andrew et al., 2017). Postprandial Burmese pythons exhibit a rapid rise in the mass of the heart and increased gene expression of muscle contractile proteins (Secor and Diamond, 2000; Andersen et al., 2005). Transcriptome analysis of Burmese python hearts revealed that 464 genes exhibited significant differential expression between the fasted and postprandial states (Wall et al., 2011). Out of a total 158 upregulated genes at 3 days after feeding compared with fasted, a half of these genes are involved in structure and movement, biogenesis, morphogenesis, structural remodeling, and organization (Wall et al., 2011). P. molurus experience postprandial increases in cardiac output due to a more than doubling of heart rate and a doubling of stroke volume (Secor et al., 2000). The heart growth in pythons occurs by cardiomyocyte hypertrophy triggered by activated PI3K/Akt/mTOR (phosphatidylinositol 3-kinase/ protein kinase B/mammalian target of rapamycin) pathway signaling (Riquelme et al., 2011) and/or by activation of the NRF2 (nuclear factor erythroid 2-related factor 2)-mediated oxidative stress response pathway (Andrew et al., 2017). Hypertrophy can promote efficient pumping by increasing the number of cardiac contractile units, while concomitantly decreasing the amount of wall stress by augmenting the wall thickness of the myocardium (Dhalla et al., 1993; Swynghedauw, 1999; Babick and Dhalla, 2007). The increased force of contraction may be the reflection of ventricular wall thickening and if this is accomplished via hypertrophy, the diffusional distance for Ca2+ will be enlarged which may require more efficient Ca2+ cycling, such as through SR (Galli et al., 2006). Corroborating these findings, our study demonstrated that postprandial pythons exhibited significantly higher cardiac contraction force and rates of force development and relaxation. These results indicate an increase in Ca2+ cycling, mainly due to a marked increase in the SERCA2a expression, enhancing cardiac muscle contractile efficiency. During diastole, the SERCA2a isoform enables quick storage of Ca2+ into the SR, which is necessary for cardiac relaxation. On the other hand, during systole, the action potential induces a minor Ca2+influx through sarcolemmal L-type Ca2+ channels, which triggers a 3

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Fig. 1. Expression levels (mean ± SEM) of key Ca2+-cycling proteins and their representative Western blotting. (A) NCX1 - Na+/Ca2+ exchanger, (B) PLB phospholamban and (C) SERCA2a - sarcoplasmic reticulum Ca2+-ATPase protein contents normalized by actin in Burmese pythons (Python molurus) from Digesting and Fasting groups (n = 6). Asterisks indicate significant differences between experimental groups (P < 0.05).

major Ca2+ release from the SR Ca2+ stores through the SR Ca2+ release channel or ryanodine receptor (RyR) (Frank et al., 2003). Consequently, Ca2+ transient amplitude is greatly dependent on the amount of Ca2+ released by the SR during ECC. Ohkusa et al. (1997) have shown that the left ventricular hypertrophy induced by pressure overload in rats is associated with the increased SR Ca2+ release and number of RyR. In transgenic mice and rats, overexpression of SERCA2a resulted in increases in the velocity of SR Ca2+ uptake and maximal rates of contraction and relaxation (Periasamy and Kalyanasundaram, 2007). In the present study, pythons from the Digesting group kept higher Fc values even at high supraphysiological stimulation frequencies (1.6 Hz or 96 bpm), resulting in a very high cardiac pumping capacity (CPC) due to improved Ca2+ handling in the postprandial period.

Maximal heart rates (fH) of Burmese pythons are approximately 25 min−1 (~0.4 Hz) at rest (Lopes et al. 2017) and 60 min−1 (~1.0 Hz) at 72 h post-feeding (Secor et al., 2000). The large metabolic response to digestion is accompanied by a doubling of heart rate and stroke volume of the heart, leading to a markedly increased (~350) cardiac output (Secor and Diamond, 1995). This is crucial to maintain adequate oxygen delivery and transport of the nutrients to several organs. In our experiments, preparations were able to contract regularly until 1.4 Hz (or 84 min−1) for fasting pythons and until 1.6 Hz (or 96 min−1), showing that the ventricular myocytes are not working in vivo at the limit of capability of the excitation-contraction cycle. In pythons, the contributing role of SR to tension generation was significantly greater following feeding. After the treatment with ryanodine, Digesting pythons showed a significant decrease in cardiac

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Fig. 2. (A) Isometric force (Fc – mN mm−2) and (B) rates (dF/dt – mN ms−1) of tension development and relaxation developed in the experimental time of 40 min by ventricular strips of P. morulus from Fasting and Digesting groups (n = 10). Mean values ± SEM. Asterisks indicate significant differences between experimental groups (P < 0.05).

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Frequency (Hz) Fig. 3. (A) Isometric force (Fc - mN mm−2), (B) rates (dF/dt - mN ms−1) of tension development and relaxation, and (C) cardiac pumping capacity (CPC mN mm−2 min−1) developed by ventricular strips of P. morulus during increases in stimulation frequency from Fasting and Digesting groups (n = 10). Mean values ± SEM. Asterisks indicate significant differences between experimental groups (P < 05). Open symbols denote a significant difference in relation to the values obtained at 0.2 Hz (P < 0.05). 5

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Frequency (Hz) Fig. 4. Role of sarcoplasmic reticulum (SR) in force generation of ventricular strips of P. morulus during increases in stimulation frequency from (A) Fasting (n = 8) and (B) Digesting groups (n = 8) in absence or presence of 10 μM ryanodine (Ry). Fc as a proportion of control is shown in C. Mean values ± SEM. Asterisks indicate significant differences between experimental groups (P < 0.05). Open symbols denote a significant difference in relation to the values obtained at 0.2 Hz (P < 0.05).

and these changes can affect its expression, giving rise to protein isoforms. For example, in transgenic mice, SERCA1a was co-localized with SERCA2a in cardiac SR (Ji et al., 1999). Cardiac myocytes expressing SERCA1a increased SR Ca2+ stores and improved the rate of Ca2+ uptake, showing increased rates of contraction and relaxation. These findings suggested that SERCA1a can functionally substitute the SERCA2a isoform in the cardiac environment, and isoform specificity is not absolute (Periasamy and Kalyanasundaram, 2007). According to Herzberg and Fernández (2002), the SERCA gene has 21 exons separated by 20 introns. This considerable number of introns forms protein variants through alternative splicing. In addition, SERCA2 protein gives rise to SERCA2b isoform with 1042 amino acid, SERCA2a with 997 amino acid (Altshuler et al., 2012) and SERCA2c with 999 amino acid (Gélébart et al., 2003). Moreover, the consumption of large prey at sporadic intervals promotes fluctuations in oxidative metabolism in pythons, resulting in an increase of up to 4000% (Andersen et al., 2005). This rapid increase in oxidative metabolism promotes large changes in metabolism gene expression. Duan et al. (2017) detected an upregulation of malate dehydrogenase, cytochromes, and ATPase-linked enzymes in Burmese python heart 48 h after ingestion of a large meal (25% of their body weight). Furthermore, mitochondrial proteins in snakes undergo an adaptive evolutionary process involving accelerated amino acid substitutions, especially in residues highly conserved in vertebrates, and

contraction force indicating that the SR makes essential contributions to ECC by regulating the Ca2+ levels in the myoplasm, mainly at low stimulation frequencies. The contribution of the SR to cytosolic Ca2+ management varies among ectotherms, ranging from 0 to > 50% depending on species (Shiels and Galli, 2014; Monteiro et al., 2017). The increased relative participation of SR to tension generation could enable the python's heart to reach maximal fH during the SDA response. Digestion caused a large increase in the heart rate of P. molurus from 19.0 ± 1.2 bpm (~0.3 Hz) to 46.2 ± 2.6 bpm (~0.8 Hz) 24 h after the snakes had been fed a meal equivalent to 23.2 ± 0.8% (means ± SE) of their body mass (Enok et al., 2012). On the other hand, during food deprivation less Ca2+ is stored in SR, probably due to the low amount of ATP available for active pumping of Ca2+ by SERCA. This metabolic condition may have contributed to a lower expression of SERCA and impaired Ca2+ handling and its role to contractile activation in the Fasting group. Impaired reuptake of Ca2+ into the SR, reflected by a reduced SERCA2a protein expression, may result in a lower SR Ca2+ concentration and intracellular Ca2+ transient (Kho et al., 2010). Regarding molecular mass of SERCA2a, there was a difference between the Digesting and Fasting groups. Periasamy and Kalyanasundaram (2007) observed that the expression of SERCA isoforms is modulated by the cardiac environment. In other words, SERCA protein can adapt to specialized functions under different conditions 6

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impairment of contractile parameters in mice ventricular myocytes (Kadambi et al., 1996). Nevertheless, in the present study, the increased PLB expression in the cardiac tissue Digesting group is not sufficiently high to inhibit SERCA2a activity. It is worth pointing out that the PLB antibody used in this study is able to recognize both phosphorylated and non-phosphorylated phospholamban. Conversely, ventricular cardiomyocytes of postprandial pythons exhibited a higher SERCA2a/PLB ratio indicating faster kinetics of contraction and relaxation due to enhanced SR function for Ca2+ handling by SERCA2a overexpression. Therefore, the balance between SERCA2a and PLB expression was able to promote the regulation of myocardial contraction and relaxation by controlling calcium transient homeostasis. The level of intracellular Ca2+ transient is tightly regulated by an equilibrium between the Ca2+ release into the cytosol and its removal by the combined action of buffers, pumps and channels (Kho et al., 2010). There was a higher expression of NCX1 in cardiac tissue of pythons from the Digestion group. This protein is the principal mechanism of Ca2+ efflux in most ectothermal vertebrates and acts on Ca2+ influx in reverse mode (Vornanen, 1996; Vornanen, 1999). In varanid lizard transsarcolemmal Ca2+ influx via reverse mode, NCX current was fourfold higher than most other ectotherms (Galli et al., 2009). However, in higher mammalian species and human myocardium, Ca2+ efflux is facilitated mainly by SERCA (70–80%) and to a lesser extent by NCX (20–30%) (Frank et al., 2003). Therefore, pythons' NCX has a high capacity for Ca2+ transport and its overexpression in the postprandial period points out its critical role in Ca2+ extrusion to relaxation, and in Ca2+ influx to contraction, allowing ventricular force maintenance during repetitive electric stimulation until the highest sustained frequency of 1.6 Hz or 96 bpm. It is known that in ectotherms the contribution of SR Ca2+ stores to contractile activation is significantly less than in endothermic hearts (Haverinen and Vornanen, 2009). Furthermore, Ca2+ handling protein expressions showed that SERCA2a, NCX1 and PLB are highly conserved since the antibodies used are specific to mammals. The high conservation and similarity are in agreement with Kimura's neutral theory (Kimura, 1983), who predicts a low rate of mutation in molecules with intense functional restriction.

A

dF/dT (mN.ms-1)

0.08

Fasting

0.04

*

* 0.00

0.2

Fasting Ry

0.4

0.6

0.8

1.0

1.2

1.4

-0.04

-0.08

Frequency (Hz)

B

dF/dT (mN.ms-1)

0.14

Digesting

0.07

* 0.00

-0.07

*

Digesting Ry

*

*

*

0.8

1.0

0.2

0.4

0.6

*

*

*

1.2

1.4

1.6

5. Conclusion After feeding, Burmese pythons display significant increases in the force of cardiac contraction. The overexpression of SERCA, PLB and NCX resulted in high efficiency of Ca2+ transport which allowed faster kinetics of contraction and relaxation as increased Ca2+ transient amplitude is a key determinant of augmented contraction force. These changes are crucial to maintaining cardiac output and a relatively high and continuous blood flow required by metabolic expenditure that occurs in the SDA response.

Frequency (Hz) Fig. 5. Rates (dF/dt - mN ms−1) of tension development (A) and relaxation (B) developed by ventricular strips of P. morulus during increases in stimulation frequency from Fasting (n = 8) and Digesting groups (n = 8) in absence or presence of 10 μM ryanodine (Ry). Mean values ± SEM. Asterisks indicate significant differences between experimental groups (P < 0.05). Open symbols denote a significant difference in relation to the values obtained at 0.2 Hz (P < 0.05).

Ethics in animal experimentation

these extreme changes in the mitochondrial proteins can extend to metabolic genes in the nuclear genome (Castoe et al., 2008, 2013). Python heart transcriptome contains of ~2800 unique genes, many of which play important roles in physiological and not pathological signaling pathways in cardiac growth (Wall et al., 2011). Since alternative splicing of SERCA genes gives multiple isoforms, changes in the metabolism of P. molurus caused by the SDA may have brought about changes in the molecular mass of SERCA2a. Ca2+-ATPase isoforms may provide additional opportunities for cardiomyocytes to tightly regulate their Ca2+ signals. PLB is a key regulator of cardiac inotropy and chronotropy (Shaikh et al., 2016). Upon phosphorylation mediated by β-adrenergic response, PLB increases SR Ca2+ pumping, because its inhibitory effect on the function of SERCA is relieved. Cardiac overexpression of PLB results in inhibition SR Ca2+ uptake, decreases in Ca2+ transient levels, and

The python care and study were conducted under the approval of the Committee of Ethics in Animal Experimentation (081/2010) from the Federal University of São Carlos and in accordance with the national guidelines for the care and use of laboratory animals. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgments This study was supported by CNPq (National Council for Scientific and Technological Development – grants #301849/2014-5 and #573921/2008-3), CAPES (Coordination for the Improvement of Higher Education Personnel) and INCT-FisComp (National Institute of 7

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Science and Technology in Comparative Physiology - FAPESP 08/ 57712-4). The authors are thankful to Mr. Angelo Carnelosi for the technical assistance, and the Jacarezário Laboratory, Department of Zoology, São Paulo State University (UNESP), Rio Claro, SP, Brazil.

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