Calcium homeostasis behavior and cardiac function on left ventricular remodeling by pressure overload

Sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and sarcolemmal Na+/Ca2+ exchanger (NCX1) structures are involved in heart cell Ca2+ homeostasis. Previous studies have shown discrepancies in their function and expression in heart failure. The goal of this study was to evaluate heart function and hypertrophied muscle Ca2+-handling protein behavior under pressure overload. Twenty male Wistar rats were divided into two groups: Aortic stenosis (AoS), induced by a clip placed at the beginning of the aorta, and Control (Sham). After 18 weeks, heart function and structure were evaluated by echocardiogram. Myocardial function was analyzed by isolated papillary muscle (IPM) at basal condition and Ca2+ protein functions were evaluated after post-pause contraction and blockage with cyclopiazonic acid in IPM. Ca2+-handling protein expression was studied by western blot (WB). Echocardiogram showed that AoS caused concentric hypertrophy with enhanced ejection fraction and diastolic dysfunction inferred by dilated left atrium and increased relative wall thickness. IPM study showed developed tension was the same in both groups. AoS showed increased stiffness revealed by enhanced resting tension, and changes in Ca2+ homeostasis shown by calcium elevation and SERCA2a blockage maneuvers. WB revealed decreased NCX1, SERCA2a, and phosphorylated phospholambam (PLB) on serine-16 in AoS. AoS had left ventricular hypertrophy and diastolic dysfunction compared to Sham; this could be related to our findings regarding calcium homeostasis behavior: deficit in NCX1, SERCA2a, and phosphorylated PLB on serine-16.


Introduction
Cardiac remodeling (CR) can be defined as changes in genetic, molecular, cellular, and interstitial expression, which can manifest as modifications in heart size, shape, and function as a response to specific aggressions, such as ischemia, inflammation, genetic mutations, and volume or pressure overload (1). CR is time-dependent and it can cause damage in the long term, representing an important risk factor for the development of ventricular dysfunction and heart failure (HF) (2,3).
An important component of cardiac muscle contraction is Ca 2+ homeostasis. In brief, this ion, which is present in the bloodstream through diet and bone balance, enters the cell through L-type channels and is stored in the sarcoplasmic reticulum until depolarization releases it to the muscle fibers through ryanodine receptors (RyR). Afterwards, with cell repolarization, the ion is recaptured through the sarcoplasmic reticulum calcium ATPase (SERCA2a) at the expense of ATP hydrolysis (13)(14)(15). Phospholambam (PLB) is a protein that regulates SER-CA2a, increasing its function when it is phosphorylated (16). The concentration of Ca 2+ in cytosol is closely controlled, and transporters such as Na + -Ca 2+ exchanger (NCX1) are very important in maintaining cell gradient.
Regarding heart dysfunction, echocardiogram studies by our research group found that 18 weeks after AoS induction, rats develop left atrium (LA) remodeling, concentric left ventricular hypertrophy, diastolic dysfunction, and systolic function maintenance, improvement, or dysfunction (1,12).
Few studies have investigated Ca 2+ -transient proteins in experimental models of heart aggression with the above features, even though they should be considered part of the mechanisms involved in cardiac function loss. Some results in this area found that SERCA2a (9,10) is reduced in AoS (11,(17)(18)(19). Also, a study using rats 6 weeks after AoS found that SERCA2a is related to functional changes (11). NCX1 was enhanced in hypertrophied hearts after infarction (20) but was at normal levels shortly after pressure overload (21). Røe et al. (22) found that rats presented enhanced SERCA and NCX1 activity after 6 weeks of aortic banding. They also concluded that LV complacency was altered regardless of changes in Ca 2+ homeostasis. Rouhana et al. (23) showed that normal SERCA and NCX1 levels after 4 weeks increased phosphorylated PLB on threonine-17; Ca 2+ was enhanced due to a lower extrusion through NCX1 and a larger leak from the sarcoplasmic reticulum through RyR. Hadipour-Lakmehsari et al. (24) reported that, after 8 weeks of transverse aortic constriction, RyR was reduced, SERCA enhanced, and PLB decreased after 2 weeks but was similar to Sham after 4 weeks.
Due to these divergent results on cardiac performance in left ventricular pressure overload remodeling and the function and expression of intracellular Ca 2+ -related proteins in non-transgenic animals, we decided to perform this study to evaluate cardiac function 18 weeks after AoS and to test whether functional variation of these proteins participates in hypertrophied muscle performance changes.

Animal care
Twenty 30-day-old male Wistar rats weighting between 60 and 80 g were obtained from the Animal Center of Botucatu Medical School, São Paulo State University, UNESP (Brazil). All experiments and procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council (1996) and were approved by Botucatu Medical School Ethics Committee (protocol FMB-PE-30/ 2013). The rats were housed 4/cage in a controlled environment (12-h light/dark cycle starting at 6:00 am) and clean-air room temperature (23°C). They received food and water ad libitum and body weight (BW) was measured weekly.

Experimental protocol
Groups. The two groups consisted of: AoS, 17 rats implanted with supravalvar bands, and Sham, 13 rats submitted to similar surgery but without band implant. During the study, 3 Sham and 5 AoS rats died. After applying the exclusion criteria below, each group contained 10 animals each.
In vitro functional study: isolated papillary muscle. Rats were euthanized 18 weeks after surgery to harvest the heart, isolated papillary muscle (IPM), and the lungs. Animals were anesthetized with sodium pentobarbital (50 mg/kg, ip), euthanized, submitted to a thoracotomy, and the hearts were quickly removed and placed in oxygenated Krebs-Henseleit solution at 28°C. Papillary muscle was dissected carefully from the left ventricle, clipped at its edges, placed vertically in a chamber containing Krebs-Henseleit solution at 28°C, oxygenated with a mixture of 0.95 O 2 and 0.5 CO 2 (pH 7.38), and stimulated with two electrodes in the solution at a rate of 0.2 Hz. Papillary muscle performance was evaluated by a technique often used in our laboratory (11,(36)(37)(38). The following basal parameters were measured from isometric contraction: peak developed tension (DT, g/mm 2 ), resting tension (RT, g/mm 2 ), time to peak tension (TPT, ms), maximum rate of tension development (+dT/dt, g/mm 2 per s), and maximum rate of tension decline ( À dT/dt, g/mm 2 per s). The mechanical behavior of papillary muscle was evaluated at baseline condition with extracellular calcium (Ca 2+ ) concentration of 2.5 mM. Then, the following inotropic maneuvers were performed: post-rest contraction, with Ca 2+ =0.5 mM; gradual Ca 2+ elevation from 0.5 to 2.5 mM, to evaluate Ca 2+ intake; and SERCA2a blockage by cyclopiazonic acid (CPA), as previously described (11,39).
The contractions were registered using a computer data system (AcqKnowledge s MP100, Biopac Systems, Inc., USA).
Papillary muscle cross-sectional area (CSA, mm 2 ) was calculated from muscle weight and length by assuming cylindrical uniformity and a specific gravity of 1.0. All force data were normalized for muscle CSA. Papillary muscles with CSA o0.5 and 41.5 mm 2 were excluded from analysis.
Post mortem morphological analysis. The presence of ventricular and atrial hypertrophy was determined by analyzing BW, tibia length (TB), and the ratio between left ventricle (LV), right ventricle (RV), atrium (AT), and total heart (TH) per TB. Water in the lungs was evaluated by measuring lung weight/TB ratio.

Statistical analysis
Data from cardiac function and morphology in vivo and in vitro, basal isometric contraction, and western blot are reported as means ± SD, and comparisons between groups were performed using the Student's t-test for independent samples. Inotropic maneuvers are reported as median, minimum, and maximum or as means±SD, using different methods of variance analysis for repeated measures. Muscle length was compared with RT and DT by the least squares method (39). The level of significance was 0.05. Normality test was performed.

Results
Post-mortem (Table 1) and echocardiographic (Table 2) structural studies showed that the AoS group had a remodeled RV and AT. The LV had a concentric hypertrophy. Echocardiographic functional analysis (Table 3) showed an improvement in systolic function (EF, FS). Diastolic functional variables did not show any change compared to Sham. However, if we consider the fact that there was an increase in relative wall thickness and LA dimensions, it is possible to infer that LV had diastolic dysfunction.
In vitro papillary muscle functional study at basal condition (Table 4) showed increased RT in AoS muscle compared to the Sham group. This behavior was also seen in the relationship between RT and muscle length variation ( Figure 1). These data showed that hypertrophied muscle is more rigid than the control, suggesting diastolic dysfunction, which corroborates the assumption   from the echocardiographic study. Systolic function remained the same in both groups (Table 4 and Figure 2), which differs from the in vivo study, where it showed an improvement in the hypertrophied heart. TPT was increased in AoS animals. Calcium elevation effects are shown in Figure 3. This maneuver revealed a significant difference between the two groups, although there is no distinction between moments in each group. AoS had significantly less response to increased extracellular Ca 2+ concentration compared to controls.
Differently from the Ca 2+ elevation maneuver, no divergence in post-rest contraction was seen between groups (Figure 4), only between different moments. Although DT and -dT/dt increased from moment 10" to 60", +dT/dt was statistically divergent after 30" in both groups.
CPA blockage (Figure 5), which is also used to analyze SERCA2a behavior, showed a difference between AoS and Sham groups for +dT/dt (2.5 mM) and -dT/dt (1.5 and 2.5 mM), which differed from the post-rest contraction study. Hypertrophied muscle presented lower contraction and relaxation speeds than control rats at these same moments in SERCA2a blockage maneuver.
To complement Ca 2+ intracellular transient analysis, we investigated the expression of proteins involved in this phenomenon. Of the studied parameters ( Figure 6), only NCX1 and SERCA2a showed decreases in hypertrophied heart compared to controls. SERCA2a/PLB and phosphorylated PLB serine 16/PLB ratios were also reduced. These data supported the hypothesis that the proteins related to intracellular Ca 2+ homeostasis were affected in hearts under pressure overload.

Discussion
It is known that when the heart is submitted to a pressure overload, such as aortic banding, its response is enhanced contractile strength, generating a thicker ventricle wall and smaller chamber diameter. In its early stages, this process helps to maintain the correct operation of the cardiovascular system, however, over time it will damage the heart and its function will fail (11,12,25).
Our echocardiography showed that after 18 weeks of aortic stenosis animals presented concentric hypertrophy, diastolic dysfunction, and increased systolic function. This result was in agreement with literature that shows enhanced systolic function after 18 weeks (12). On the other hand, Mendes et al., using the same methodology and observation time, found an initial decrease after 18 weeks (1) in AoS. Although our study found no statistical difference between the variables that analyze diastolic function in echocardiogram, the increase in relative wall thickness implies in hypertrophy, which impairs muscle energy balance, jeopardizing diastole, and increasing end diastolic pressure, which ends up leading to left atrium enlargement, also found in our study. Literature shows that diastolic dysfunction is usually found in this experimental cardiopathy model after 6 weeks of aortic stenosis (11,12,25).   We studied isolated papillary muscle to verify whether the alterations in hypertrophied muscle seen in vivo were still present even under controlled conditions such as in vitro preload, afterload, and heart rate. We also analyzed whether the calcium transient misbalance could be one of the mechanisms involved in this experimental heart failure model.
The data were partially compatible with echocardiogram. RT was elevated, both at basal condition and during muscle length variation, proving that AoS animals had lower heart muscle complacency, which indicates diastolic dysfunction. According to Silveira et al. (11), one factor that could explain this finding is an increase in cytosolic Ca 2+ at the end of the diastole, leaving ions bound to C troponin.    However, although the image exam showed enhanced systolic function, DT in this study was similar in both groups. Higher TPT in AoS could indicate the beginning of mechanical systolic dysfunction, which was not detected in the echocardiogram. This behavioral distinction is probably due to the fact that papillary muscle is analyzed in situations with controlled load and heart rate and is free from neurohumoral variations, present in in vivo heart.
The inotropic maneuvers used in our papillary muscle study showed that dysfunctional hypertrophied myocardium may be related to calcium-handling alterations. Thus, elevated extracellular calcium concentrations displayed a robust difference in behavior between groups, with aortic stenosis animals presenting less response to an increase in this ion. This pronounced change did not appear in postrest contraction maneuver but was present at a lower level in SERCA2a blockage with cyclopiazonic acid. These results suggested a dysfunction in Ca 2+ entry through L-type Ca 2+ channels, although expression was normal in the molecular study. There was also less damage of the intracellular Ca 2+ cycle in the myocardium, which could be compatible with decreased SERCA2a and SERCA2a/PLB expression.
The decrease in NCX1 expression, the most important mechanism involved in calcium efflux across the plasma membrane, would allude to enhanced intracellular Ca 2+ in hypertrophied muscle, which corroborates the idea that remaining ions cause the diastolic dysfunction shown in IPM through an elevation in resting tension.
An interesting review looked at Ca 2+ -handling proteins and their relationship with hypertrophied muscle (40). In several studies, Locatelli et al. (40) found that NCX1 is usually enhanced in heart failure and hypertrophy and that SERCA expression is reduced or regulator levels are increased. This review only corroborates all the divergences between studies and between data within studies that are cited in the present article. Perhaps variables such as similarity between animals, experiment conditions, and evaluator performances have a huge influence on results; that is why scientific research needs to continue in its quest for an answer.
In conclusion, data in our study showed that 18 weeks after AoS surgery rats presented diastolic dysfunction, which could be related to changes in calcium-homeostasis behavior.