Myocardial Dysfunction after Severe Food Restriction Is Linked to Changes in the Calcium-Handling Properties in Rats

Severe food restriction (FR) impairs cardiac performance, although the causative mechanisms remain elusive. Since proteins associated with calcium handling may contribute to cardiac dysfunction, this study aimed to evaluate whether severe FR results in alterations in the expression and activity of Ca2+-handling proteins that contribute to impaired myocardial performance. Male 60-day-old Wistar–Kyoto rats were fed a control or restricted diet (50% reduction in the food consumed by the control group) for 90 days. Body weight, body fat pads, adiposity index, as well as the weights of the soleus muscle and lung, were obtained. Cardiac remodeling was assessed by morphological measures. The myocardial contractile performance was analyzed in isolated papillary muscles during the administration of extracellular Ca2+ and in the absence or presence of a sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) specific blocker. The expression of Ca2+-handling regulatory proteins was analyzed via Western Blot. Severe FR resulted in a 50% decrease in body weight and adiposity measures. Cardiac morphometry was substantially altered, as heart weights were nearly twofold lower in FR rats. Papillary muscles isolated from FR hearts displayed mechanical dysfunction, including decreased developed tension and reduced contractility and relaxation. The administration of a SERCA2a blocker led to further decrements in contractile function in FR hearts, suggesting impaired SERCA2a activity. Moreover, the FR rats presented a lower expression of L-type Ca2+ channels. Therefore, myocardial dysfunction induced by severe food restriction is associated with changes in the calcium-handling properties in rats.

Calcium handling is an essential process that facilitates myocardial contraction and relaxation. Previous work examined whether alterations in Ca 2+ handling participates in the decline of myocardial function in response to malnutrition, but the results are divergent [12][13][14][15][16][17]29,30]. While one study suggested that cardiac ultrastructural changes were responsible for deleterious cardiac outcomes [17], several studies demonstrated that abnormal activity of Ca 2+ handling specific regulatory proteins has a fundamental role in the negative repercussions of severe FR on myocardial function [13,15,16,19]. Although malnutrition was shown to alter the gene transcription process that forms proteins involved in Ca 2+ handling [14,15,19,31], limited studies evaluated changes at the protein level. Work by De Tomasi et al. [15] focused on the L-type calcium channel, and found only a significant reduction in its protein expression in the hearts of FR rats. However, as it is known that the levels of transcripts and proteins are not always in direct correlation [32,33] and no study evaluated all the proteins involved in Ca 2+ handling, additional work is required to understand the role that this mechanism plays in the development of cardiac dysfunction during long-term FR.
Thus, the aim of this study is to test the hypothesis that malnutrition induced by restricting the food intake causes the deterioration of myocardial function due to alterations in the expression of calcium-handling proteins and sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) activity. The results of our study demonstrate that 90 days of FR severely diminishes cardiac function in rats, which is in part due to decreased intracellular calcium influx through the L-type channel and depressed SERCA2a activity.

Animal Model and Experimental Protocol
Sixty-day-old male Wistar-Kyoto rats were obtained from UNICAMP-the State University of Campinas, Brazil. The animals were randomized into control (C) and food restriction (FR) groups. C animals (n = 14) received commercial chow (3.76% fat, 20.96% protein, 52.28% carbohydrate, 9.60% fiber, and 13.40% moisture) and water ad libitum. The FR group (n = 13) was subjected to a severe food restriction equivalent to 50% of the average amount consumed by group C. Food intake for the control group was measured daily and used to calculate food quantity for the FR group. Rats were maintained on this dietary regimen for 90 days and were weighed once a week. The rats were kept in individual cages at a 23 • C room temperature, with light cycles of 12 h and relative humidity of 60%. At the end of experimental protocol, the animals were anesthetized with an intramuscular injection of ketamine (50 mg/kg; Dopalen ® , Sespo Indústria e Comércio Ltd.a-Divisão Vetbrands, Jacareí, São Paulo, Brasil) and xylazine (10 mg/kg; Dopalen ® , Sespo Indústria e Comércio Ltd.a-Divisão Vetbrands, Jacareí, São Paulo, Brasil), decapitated, and thoracotomized.
The project was approved by the "Committee for Experimental Research Ethics of the Faculty of Medicine in Botucatu-UNESP" (CEUA 755-2009), in accordance with the "Guide for the Care and Use of Laboratory Animals".

General Characteristics
Initial (IBW) and final body weights (FBW), total body fat (BF), adiposity index (AI), naso-anal and tibia length, and soleus muscle and lung weight were measured to assess the effects of food restriction on body parameters of rats. The adipose tissue fat pads (epididymal, retroperitoneal, and visceral) were dissected and weighed. Adiposity index (AI) was calculated using the formula: AI = [total body fat (BF)/FBW] × 100. BF was measured from the sum of the individual fat pad weights as follows: BF = epididymal fat + retroperitoneal fat + visceral fat.

Cardiac Morphological Post Mortem Study
Cardiac remodeling was determined by macroscopic analysis of the following parameters: total weight of the heart (HW) and left ventricle (LVW), right ventricle (RVW), and atria (ATW), and normalized to body weight.

Statistical Analysis
All the data were tested for normality before statistical analysis using the Shapiro-Wilk test. Data from physical characteristics, cardiac post-mortem morphology, and Western blot of calcium-handling proteins were reported by descriptive measures of position and variability and subjected to the "t" test for independent samples. For isolated papillary muscle experiments, the adjustment of the DT, +dT/dt, and −dT/dt response linear model, in the presence and absence of CPA as a function of the extracellular Ca 2+ concentration elevation, expressed by Response Variable = a + b/[Ca 2+ ] extracellular , was performed by the minimum squares technique complemented with the comparative test of average profiles of responses regarding the parallelism and coincidence of the adjusted models to the group. The statistical analyses were performed using SigmaPlot 12.0 (Systat Software, Inc., San Jose, CA, USA). The level of significance for all variables was p < 0.05.

Physical Characteristics
Severe food restriction (FR) resulted in substantial changes to the physical phenotype of the rats. As shown in Table 1, 90 days of FR significantly reduced body weight and fat mass measures, resulting in an approximate 12-fold decrease in the adiposity index. Furthermore, masses of the soleus muscle, lung, and tibia were significantly lower in FR rats. These results show that 90 days of FR severely alters physical phenotype, including the impairment and/or regression of organ growth.

Macroscopic Cardiac Morphology
As shown in Table 2, the weight of the left ventricle (LV), right ventricle (RV), and atria (AT) were approximately 50% lower in FR rats compared to their controls. However, the data normalized by FBW were similar between the groups.

Assessment of Papillary Muscle Function
To determine whether abnormal heart size was associated with myocardial dysfunction, the contractile performance of LV papillary muscles isolated from control and FR hearts was measured. In response to increasing extracellular Ca 2+ concentration, the developed tension (DT) was significantly reduced in papillary muscles from FR hearts ( Figure 1A). Papillary muscles from FR hearts also had significantly lower values for positive tension derivative (+dT/dt) and negative tension derivative (−dT/dt) ( Figure 1B,C), which is consistent with impaired contractility and relaxation. Additionally, the administration of cyclopiazonic acid (CPA), which is a specific inhibitor of SERCA2a, further decreased +dT/dt and −dT/dt ( Figure 1B,C) in FR hearts. Moreover, Figure 2 shows that the blocking percentage by CPA was lower in the FR than the control group. This result suggests a prior impairment of SERCA2a activity under caloric restriction, which would reduce the magnitude of the effect of cyclopiazonic acid inhibition. Overall, these findings show that 90 days of FR severely impairs myocardial contraction and relaxation, which is likely due to depressed SERCA2a activity.

Expression of Calcium-Handling Protein
Calcium-handling proteins are integral in facilitating myocardial contraction and relaxation. Therefore, a Western blot analysis was conducted focusing on the primary proteins involved in calcium handling, including SERCA2a, phospholamban (PLB), ryanodine receptor (RYR), calsequestrin (CSQ), sodium-calcium exchanger (NCX), and the L-type calcium channel (L channel). As shown in Figure 3B,F-H, no statistical differences were noted in SERCA2a, RYR, CSQ, or NCX. Additionally, no differences in PLB or the phosphorylation sites at PLB Ser 16 or PLB Thr 17 were observed ( Figure 3C-E).

Discussion
The purpose of this study was to investigate the contribution of alterations in the expression of Ca 2+ -handling proteins and SERCA2a activity to myocardial performance during severe food restriction. The nutritional protocol used in this study was sufficient in duration and intensity to cause malnutrition, which is in agreement with previous studies [12,13,17]. The main findings are that malnutrition reduces the myocardial L-type calcium channels protein expression and alters SERCA2a activity, as expressed by the decrement of the mechanical responses to extracellular Ca 2+ elevation in the absence and presence of cyclopiazonic acid. Overall, these changes in the calcium-handling capacity of the cardiac myocyte are critical in the development of myocardial dysfunction during severe food restriction.
In agreement with previous literature [11][12][13][14][15]17,19,31], the 50% reduction of food intake used in this study promoted drastic changes in the body composition of animals, as visualized by a reduction in all of the variables related to fat and body weight. Malnutrition causes damage to several segments of the body system, including a substantial reduction of hind limb muscles such as the soleus, plantaris, gastrocnemius, and extensor digitorum longus muscles [12,13,15,37,38]. In addition, food restriction leads to severe changes in the cardiorespiratory structures, particularly the weights of the lungs, atria, and ventricles [13,14,16]. These above responses, which were also observed in our study, are consistent with increased protein catabolism, as well as decreased protein synthesis, since the restriction of pellet intake produces protein and energy deficiency [38].
In isolated papillary muscle analysis, malnutrition resulted in decreased contractile (DT and +dT/dt) and relaxation (−dT/dt) capacity, which is in accordance with functional impairments observed in precedent studies [12,13,19,21,22,24,39]. At the molecular level, the quantity and quality of contractile proteins and the availability of energy and Ca 2+ are crucial factors in the regulation of myocardial performance [40,41]. Previous work from our laboratory showed that severe food restriction causes a reduction of myofibril density and myofilament disorganization [12,17,20,26]. Furthermore, the authors reported myosin isoform remodeling with the down-regulation of alpha-(V 1 ) and up-regulation of beta-myosin heavy chain (V 3 ) genes, which present faster and lower ATPase activity, respectively [24,30]. In addition, the constant low availability of substrates during the malnutrition period may lead to mitochondrial injury [17,20], leading to a lack of ATP production, which may impair the capacity of the ATP-dependent proteins.
The primary aim of this study was to evaluate the role of Ca 2+ -handling proteins in the development of myocardial dysfunction induced by malnutrition. Myocardial Ca 2+ -handling inadequacies are among the most critical responses to cardiac aggression [12][13][14][15][16][17]19,[29][30][31]. Previous studies reported alterations in the activity [13,15,16] and content [14,15,31] of Ca 2+ -handling regulatory components, which is consistent with our current observations. With any cardiac remodeling process at the macro or micro level, the initial intent of adaptations in Ca 2+ handling is to attenuate the insult imposed on the myocardium in an effort to maintain the functional balance. However, these adaptations are incapable of enduring the aggression over time, contributing to the establishment of the dysfunctional condition [42,43]. In the present study, 90 days of severe food restriction resulted in alterations in SERCA2a activity and, among the analyzed proteins, only a reduction in L-type calcium channels protein expression. During the Ca 2+ transient, the SERCA2a is an ATP-dependent protein that is responsible for the reuptake of Ca 2+ into the sarcoplasmic reticulum of the cardiomyocyte, which is crucial for the maintenance of the cytosolic concentration. Although we did not observe a reduction of SERCA2a protein expression, impaired activity was noted in the papillary muscle assay in the presence of cyclopiazonic acid, as noted previously [19]. This finding is likely due to the low-energy capacity in severe food restriction, which limits the ATP required for proper SERCA2a function. Impaired SERCA2a activity may provoke the elevation of diastolic Ca 2+ , which would lead to the increased importance of the exchanger Na + /Ca 2+ (NCX) cytosolic Ca 2+ extrusion. Since the NCX mechanism has a reduced capacity to remove cytosolic Ca 2+ , alterations in Ca 2+ handling persist.
In disagreement with our hypothesis, in which changes in several Ca 2+ -handling proteins were expected, our study showed only reduction in the expression of the L-type calcium channel protein due to food restriction, which is in accordance with a previous report [15]. Although the reason for this outcome is unclear, we speculate that this may be an adaptive response, serving as a cellular defense mechanism in response to dysfunctional SERCA2a activity. In this regard, myocyte Ca 2+ influx is decreased in order to avoid Ca 2+ accumulation, particularly during diastole. However, the reduction of Ca 2+ influx across the plasma membrane as a result of decreased L-type calcium channel expression over time may lead to impairments in cardiomyocyte force development, as evidenced by reduced values of DT and +dT/dt in animals suffering from malnutrition. Although the functional control of the L-type channel is well described, little is known about the processes involved in the expression of this sarcolemma channel. The L-type channel is composed of three subunits (α 1C , α 2 δ, and β) [44], with the α 1C pore-forming subunit analyzed in this study. The expression of the α 1C subunit protein is regulated by β and α adrenergic systems [45], angiotensin II [46], and calcium flux through the L-type channel [47][48][49]. Based on our results, it is possible that the cytosolic Ca 2+ "leftover", due to SERCA2a dysfunction, activated the auto-regulation mechanism of L-type channel protein expression on the distal carboxyl-terminus via a process sensitive to Ca 2+ flux alterations [47][48][49]. In conditions in which there is undue cytosolic Ca 2+ accumulation, the hyperactivation of this regulatory mechanism may be related to the decrease in the protein expression of these channels. For better visualization of the food restriction effects over the myocardium, we constructed a figure placing the main molecular alterations and suggested a hypothesis that attributed cardiac dysfunction to malnutrition, as can be seen in Figure 4. Overview of food restriction effects over myocardium and presumable interactions between molecular changes and myocardial mechanical impairment. Food restriction reduced sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) activity and L-type calcium channel (L channel) expression and impaired myocardial mechanical performance. Hypothetical adenosine triphosphate (ATP) depletion due to malnutrition could reduce the SERCA2a activity, leading to a high amount of residual cytosolic calcium. In turn, residual calcium signals to deplete the release of calcium by the ryanodine receptor (RYR), which is achieved by the reduced calcium entry secondary to the decreased protein expression of the L channel. This sequence of events involving the myocardial calcium handling would be a plausible mechanism by which the food restriction deteriorates the cardiac function. Ca 2+ , calcium; Na + , sodium; NCX, sodium-calcium exchanger; SR, sarcoplasmic reticulum; CSQ, calsequestrin; PLB, phospholamban; DT, developed tension; +dT/dt, positive tension derivative; −dT/dt negative tension derivative. Therefore, our results show that malnutrition, due to 90 days of severe food restriction, leads to cardiac dysfunction. The deterioration of myocardial function may be the consequence of a reduction of L-type calcium channels protein expression and impaired SERCA2a activity. Future studies are required to understand the mechanisms that cause inadequacies in L-type Ca 2+ calcium channels and SERCA2a activity during severe food restriction, which ultimately contribute to malnutrition-induced myocardial dysfunction.