Roles of sarcoplasmic reticulum Ca2+ ATPase pump in the impairments of lymphatic contractile activity in a metabolic syndrome rat model

The intrinsic lymphatic contractile activity is necessary for proper lymph transport. Mesenteric lymphatic vessels from high-fructose diet-induced metabolic syndrome (MetSyn) rats exhibited impairments in its intrinsic phasic contractile activity; however, the molecular mechanisms responsible for the weaker lymphatic pumping activity in MetSyn conditions are unknown. Several metabolic disease models have shown that dysregulation of sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump is one of the key determinants of the phenotypes seen in various muscle tissues. Hence, we hypothesized that a decrease in SERCA pump expression and/or activity in lymphatic muscle influences the diminished lymphatic vessel contractions in MetSyn animals. Results demonstrated that SERCA inhibitor, thapsigargin, significantly reduced lymphatic phasic contractile frequency and amplitude in control vessels, whereas, the reduced MetSyn lymphatic contractile activity was not further diminished by thapsigargin. While SERCA2a expression was significantly decreased in MetSyn lymphatic vessels, myosin light chain 20, MLC20 phosphorylation was increased in these vessels. Additionally, insulin resistant lymphatic muscle cells exhibited elevated intracellular calcium and decreased SERCA2a expression and activity. The SERCA activator, CDN 1163 partially restored lymphatic contractile activity in MetSyn lymphatic vessel by increasing phasic contractile frequency. Thus, our data provide the first evidence that SERCA2a modulates the lymphatic pumping activity by regulating phasic contractile amplitude and frequency, but not the lymphatic tone. Diminished lymphatic contractile activity in the vessels from the MetSyn animal is associated with the decreased SERCA2a expression and impaired SERCA2 activity in lymphatic muscle.

Insulin resistance is one of the major causes of metabolic syndrome (MetSyn) or related metabolic disorders that are associated with an enormous health burden worldwide 1 . MetSyn is now one of the most prevalent diseases globally and increases the risk for all causes of mortalities, including cardiovascular diseases 1,2 . Clinical studies have established the link between obesity and lymphatic dysfunction, which is associated with increased susceptibility for developing lymphedema [3][4][5][6] . Mice heterozygous for Prox1, a master lymphatic endothelial transcription factor, consistently develop adult onset obesity coupled with increased chyle accumulation in the thoracic cavity 7,8 . In addition, these mice exhibited higher leptin and insulin levels 8 , which are pathological determinant factors of insulin resistance suggesting a direct role of the lymphatic system in metabolic dysfunction. We have previously reported that a high-fructose-fed rat model of MetSyn presented a significant reduction in lymphatic pumping as a consequence of decreased phasic contractile frequency and impaired intrinsic lymphatic muscle force production 9,10 . These findings have been corroborated in the obese mouse models that diminished pressureinduced frequency in collecting lymphatic vessels 11 . We have also demonstrated that insulin resistance directly impaired cellular bioenergetics and decreased the relative levels of the regulatory molecule, myosin light chain Scientific RepoRtS | (2020) 10:12320 | https://doi.org/10.1038/s41598-020-69196-4 www.nature.com/scientificreports/ 20 (MLC 20 ) in lymphatic muscle cells (LMCs). However, the direct mechanisms that reduce lymphatic pumping activity in the MetSyn animals have not been completely understood. The active spontaneous pumping of lymphatics is achieved by the intrinsic contractile activity of the lymphatic muscle cells in the wall of collecting lymphatic vessels that produces the rhythmic phasic contractions. The lymphatic muscle cells exhibit unique characteristics similar to both vascular smooth muscle and cardiac muscle cells 12 . Like vascular smooth muscle, lymphatics show the contractile activity that is regulated by various vasoactive (e.g., substance p, endothelin-1, histamine, acetylcholine, etc.) and mechanical factors (e.g., transmural pressure, flow, etc.). In addition, lymphatic muscle displays a rapid phasic contraction that is mainly achieved by the intrinsic pumping characteristics. While the resting membrane potential is mediated by Cl −13 and voltage gated K + channel 14 , lymphatic contractions are predominantly regulated by Ca 2+ influx 15,16 . The intracellular Ca 2+ concentration determines the lymphatic vessel contraction and similar to most other smooth muscle types, Ca 2+ binds to calmodulin to form an active Ca 2+ /calmodulin complex, which activates myosin light chain kinase, a key regulatory molecule that phosphorylate MLC 20 [17][18][19][20] . Since MLC 20 phosphorylation is increased in the insulin resistant LMCs 21 , we propose that regulatory molecules of Ca 2+ and/or Ca 2+ homeostasis are impaired in the MetSyn lymphatics.
The endoplasmic reticulum (ER) is the main storage site of intracellular Ca 2+ that maintains intracellular Ca 2+ levels ~ 10,000-fold lower than extracellular and ER Ca 2+ concentrations 22,23 . Re-uptake of Ca 2+ into the ER by sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) is necessary for muscle relaxation and restores ER Ca 2+ levels for subsequent systolic and diastolic cycles followed by transiently increased intracellular Ca 2+ levels. Alterations in Ca 2+ homeostasis have been shown to trigger lymphatic dysfunction. When L-type Ca 2+ channels were disrupted, stretch-induced lymphatic contractile amplitude was diminished; whereas, T-type, 'transient, ' Ca 2+ channel inhibition reduced the stretch-induced phasic contractile frequency in the lymphatics 24 . Disrupting ER Ca 2+ in isolated bovine lymphatic vessels caused lymphatic dysfunction 25 . Additionally, SERCA2 activity and expression are diminished in vascular smooth muscle 26,27 and heart 28,29 in different animal models of obesity/ diabetes, highlighting a potential pathological role for SERCA2 dysfunction and disturbed intracellular Ca 2+ homeostasis in the development of metabolic abnormalities in insulin resistance and diabetes. However, the role of SERCA2 in lymphatic pumping activity and possible pathophysiological roles in MetSyn have not yet been examined.
Our previous data showed negative chronotropic effects at all transmural pressures that effectively reduced the intrinsic flow generating capacity of mesenteric lymphatic vessels in MetSyn rats 9,10 . Additionally, insulin resistance increased MLC 20 phosphorylation in LMCs 21 that is mediated by intracellular Ca 2+20 . Hence, we hypothesized that a decrease in SERCA expression and/or activity in lymphatic muscle influences Ca 2+ homeostasis in LMCs, and consequently, diminishes lymphatic contractile activity in MetSyn animals. To test this hypothesis, we assessed the expression of SERCA2 isoforms in lymphatic muscle and determined the role of SERCA2 in the regulation of lymphatic contraction in the normal and MetSyn conditions.

Results
MetSyn rats exhibit an altered body composition by decreasing skeletal muscle mass, but increasing body fat deposition and cardiac muscle mass. We have previously reported hyperinsulinemia and hyperlipidemia in high-fructose-induced MetSyn animals 9 . As we expected, blood glucose levels in the high-fructose diet fed rats were significantly increased (7.37 ± 0.39 vs. 13.37 ± 0.55 mM, control vs. MetSyn, p < 0.05) compared with the control group rats in the normal-diet chow ( Table 1). As we reported in our previous studies, we did not observe any significant increase in body mass over the 7-10 weeks diet period in MetSyn rats comparing to the control group. However, skeletal muscle mass was found to be decreased in the MetSyn group when compared to the controls, which is consistent with the common pathological phenotype in metabolic disorders 30 . Soleus and tibialis anterior (TA) muscles from MetSyn rats showed a significant decrease in muscle mass (p < 0.05) and in the fiber type distribution that is skewed to the smaller muscle fiber (Fig. 1A-D). In contrast, accumulation of body fat in both visceral and inguinal subcutaneous fat pad was significantly higher in MetSyn rats (Table 1). Additionally, heart weight was significantly increased in MetSyn animals and exhibited enlarged cardiac myocytes comparing with the myocytes from control rats (Table 1 and Fig. 1E, F).  31 . At concentrations higher than 5 μM, thapsigargin completely inhibited lymphatic vessels phasic contractions (data not shown). Therefore, we examined the effects of thapsigargin in the dose range of 500 nM to 2 μM on lymphatic vessel contractile properties. While 500 nM dose of thapsigargin did not affect the contractile frequency, amplitude, ejection fraction and fractional pump flow at any tested transmural pressures, 1.0 μM thapsigargin significantly reduced the contraction amplitude and the fractional pump flow at P = 3 or P = 5 cmH 2 O (Fig. 2C,D). Though the contractile frequency of the lymphatics was not significantly reduced when treated with   Fig. 2C,D). 2 μM thapsigargin was found to severely impair the lymphatic contractions with decreased frequency and amplitude, and thus fractional pump flow ( Fig. 2B-D). Thapsigargin did not affect lymphatic vessel tone at all tested transluminal pressures (Fig. 2E). Therefore, we employed 1.5 μM of thapsigargin to examine the role of SERCA activity in the regulation of lymphatic vessel contractility of MetSyn animals.    www.nature.com/scientificreports/ p < 0.05, Fig. 3B,C, Table 2), as we have previously reported 9,10 . However, thapsigargin did not further decrease the contractile frequency of lymphatic vessels from MetSyn animals. In addition, the reduction in contractile frequency due to thapsigargin in control animals was found to be similar to the contractile frequency in MetSyn animals (Fig. 3C). Contraction amplitude was not significantly different among control and MetSyn lymphatic vessels (Fig. 3D). Thapsigargin diminished contraction amplitude significantly only in control lymphatic vessels, not in MetSyn lymphatic vessel though it showed a decrease trend (Fig. 3D). Additionally, thapsigargin significantly reduced the ejection fraction in both control and MetSyn animals at transmural pressures, P = 1, 3 and 5 cmH 2 O, while there was no significant difference between the control and MetSyn groups ( Table 2). The fractional pump flow were also reduced significantly by thapsigargin in control lymphatic vessels in all transmural pressures (p < 0.05, Fig. 3E). While fractional pump flow were significantly decreased in the lymphatic vessels from MetSyn rats compared to the control group, thapsigargin did not further diminish these parameters in MetSyn group (Fig. 3E). Thapsigargin did not affect lymphatic tone both in control and MetSyn lymphatic vessels (Fig. 3F). A summary of all the lymphatic contractile parameters is given in Table 2.
Decreased SERCA2a expression in MetSyn lymphatic vessel. To assess the expression of SERCA, we performed immunofluorescence analyses on isolated mesenteric lymphatic vessels from control and MetSyn rats using striated muscle-specific SERCA2a or striated and smooth muscle isoform, SERCA2b specific antibody co-stained with α-smooth muscle actin (α-SMA) antibody. Both SERCA2a and SERCA2b positive staining were revealed on the lymphatic vessel wall. SERCA2a and SERCA2b are co-stained with α-SMA in lymphatic muscle, indicating both SERCA2a and SERCA2b are present in lymphatic muscle (Fig. 4A,C). Negative controls were performed with normal rabbit IgG (data not shown). Further quantitative analyses showed that SERCA2a expression was significantly decreased in the MetSyn lymphatic muscle compared to control group (0.56 ± 0.39 fold, p < 0.001, n = 9 vessels from three animals/group, Fig. 4A,B); however, there was no significant differences in the SERCA2b expression among the control and MetSyn groups. We further examined the relative levels of MLC 20 phosphorylation in the control and MetSyn lymphatic vessels. MetSyn lymphatic vessels displayed significantly higher levels of phosphorylated MLC 20 (p < 0.008, Fig. 4E,F).

Insulin resistance impaired SERCA2 activity in LMCs.
To address whether insulin resistance conditions in LMCs impair SERCA activity and expression, and calcium regulation, LMCs were cultured in hyperglycemia and hyperinsulinemia conditions for 48 h as described in our previous study 21 . Insulin resistant LMCs showed elevated basal intracellular Ca 2+ levels (92.1 ± 2.64 nM) compared to controls (77.19 ± 1.01 nM, p < 0.001) and other groups (Fig. 5A,B). We used 5 μM of thapsigargin that prevented Ca 2+ uptake into the endoplasmic reticulum by blocking SERCA. Thus, inhibiting SERCA contributed to significantly increase peak intracellular Ca 2+ in LMCs. Peak intracellular Ca 2+ and transient time to reach the peak Ca 2+ levels were not significantly different between the groups (Fig. 5A). The amplitude between basal and peak Ca 2+ levels in the presence of the thapsigargin was found to be significantly decreased in insulin resistant LMCs (Control, 52.36 ± 2.59 nM vs. HG + Insulin, 30.52 ± 2.42 nM, p < 0.001), indicating decreased SERCA activity in these cells (Fig. 5A,C). No difference was observed between groups in the peak Ca 2+ levels when we depolarized the cells using 80 mM K + (data not shown). ER specific SEC61 protein staining showed no differences in the ER morphology between the groups. SERCA2a expression was significantly lower in insulin resistant LMCs when compared to control LMCs (− 0.41 fold vs. control, p < 0.032) (Fig. 5D,E). Corroborating our immunofluorescence data, western blot analyses demonstrated that SERCA2a protein expression was decreased in insulin resistant LMCs (− 0.58 fold vs. control, p < 0.026) while SERCA2b expression remained unchanged (Fig. 5F,G). Next, we tested whether increased extracellular free calcium level in LMCs would directly influence MLC 20 phosphorylation levels as external calcium levels affect intracellular calcium level and muscle contractility [32][33][34] . LMCs were grown in the media containing different free calcium concentration solution (calcium free to pCa3.5). MLC 20 phosphorylation was found to be increased in LMCs grown under increasing extracellular Ca 2+ concentrations and showed significant increase at pCa 6.5 or at higher pCa (p < 0.05, Fig. 5H).

Activation of SeRcA pump partially improves lymphatic contractile activity in MetSyn animals.
CDN1163 is a small molecule that activates SERCA2 by directly binding to the SERCA2 structure and increases SERCA2 V max activity [35][36][37] . In this study, we tested whether exogenously adding CDN1163 in the isolated lymphatic vessel preparations of MetSyn rats would improve its pumping activity. We selected different doses of CDN1163 (1, 5, and 10 μM) based on previous studies. We initially determined the effects of CDN1163 on lymphatic contractile parameters of the control vessels. While lymphatic contractile frequency was not affected in the presence of 1 μM CDN 1163, 5 μM CDN1163 significantly increased contractile frequency of lymphatic vessels at transmural pressures, 3 cmH 2 O (17.74 ± 2.7 contractions/min, p < 0.022) and 5cmH 2 O (18.16 ± 1.9 contractions/min, p < 0.016, Fig. 6B). Lymphatic contractile frequency was increased at all different transmural pressures in the presence of 10 μm CDN1163 (p < 0.001, Fig. 6B). Though CDN1163 did not alter significantly the contractile amplitude and tone (Fig. 6C,E), there was a trend showing a decrease in lymphatic contraction amplitude in a dose dependent manner (Fig. 6C). Ejection fraction was not significantly affected in the presence of CDN1163 (Table 2). Therefore, even if CDN1163 significantly increased contractile frequency of control lymphatic vessels, lymphatic fractional pump flow did not change at all different transmural pressures in the presence of 5 μm CDN1163 (Fig. 6D). Hence, we used 5 μm CDN1163 that showed positive chronotropic effect without negative inotropic effect (Fig. 6A-E), to test whether CDN1163 ameliorates MetSyn induced impaired lymphatic contractile activity. CDN1163 significantly improved contractile frequency of lymphatic vessels from MetSyn animals, as we found in control vessels (Fig. 6F-H). Similar to lymphatic vessels from control animals, SERCA activation did not improve fractional pump flow significantly in the lymphatic vessels from MetSyn www.nature.com/scientificreports/ group (Fig. 6J). Though there was a tendency of reduction in contraction amplitude by CDN1163 on both control and MetSyn lymphatic vessels, the differences were not significant (Fig. 6I). Additionally, we do not see any noticeable difference in lymphatic tone by CDN 1163 in both control and MetSyn lymphatic vessels (Fig. 6K).
The other calculated lymphatic contractile parameters of control and MetSyn rats are detailed in Table 2.

Discussion
The data presented in this study provide the first evidence that the striated muscle-specific, SERCA2a pump that is present in lymphatic muscle modulates the lymphatic pumping activity by regulating phasic contractile amplitude and frequency, but not the lymphatic tone. Additionally, diminished lymphatic contractile activity in the vessels from the MetSyn animal is associated with the decreased SERCA2a expression and SERCA2 dysfunction. SERCA activator, CDN1163 significantly improved the contractile frequency and partially restored lymphatic pump function in MetSyn mesenteric lymphatic vessels. Additionally, our data demonstrate that reduced SERCA2a expression resulted in impaired Ca 2+ homeostasis in insulin resistant LMCs. Collectively, these data suggest MetSyn conditions diminished SERCA2a expression and SERCA activity in lymphatic muscle, consequently reduced lymphatic contractile activity in MetSyn rats (Fig. 7).
In keeping with previous studies, we found that the MetSyn animals had increased glucose levels and other characteristics of MetSyn conditions, such as elevated levels of triglycerides and cholesterol 9,38,39 . Further, we observed increased subcutaneous and visceral body fat with decreased muscle mass (Table 1 and Fig. 1). Cross sectional analysis of the TA and soleus muscle from MetSyn animals showed left skewed in fiber type size distribution graph, indicating muscle atrophy (Fig. 1C,D). The average cross-sectional area was significantly smaller in both MetSyn TA and soleus muscle (Fig. 1A-D). The muscle atrophy in metabolic diseases is defined by the pathological term 'sarcopenic obesity' [40][41][42] , and altered body composition signifies the important pathological aspects of our MetSyn model. In addition, the muscle loss in obesity or metabolic disease could result from chronic inflammation [43][44][45] . Our previous data showed increased proinflammatory signaling in MetSyn mesentery bed with activation of M1 macrophages 10 . In addition, dietary endotoxin, LPS, that also cause insulin resistance, altered inflammatory immune response in the lymphatic mesenteric bed 46 . Hence, we speculate that muscle atrophy in fructose diet-induced MetSyn rats could be due to systemic inflammatory effects. Cardiac hypertrophic remodeling is one of the prevailing pathological features of metabolic disorders due to chronic systemic inflammation [47][48][49] . Our data showed that hearts from fructose-induced MetSyn rats displayed increased heart weight and enlarged cardiomyocyte cross-sectional area (Table 1 and Fig. 1).
SERCA is one of the key regulators of striated and smooth muscles' contractions by regulating intracellular Ca 2+ levels 50,51 . While the role of SERCA in cardiac, skeletal, and smooth muscle have been largely investigated, very little is known about the role of SERCA in lymphatic muscle. One study showed that a SERCA inhibitor, cyclopiazonic acid (7 μm) completely blocked lymphatic function in bovine mesentery lymph vessels at all different transmural pressures 25 . However, whether the inhibitory effects of SERCA was due to chronotropic or inotropic effects had not been tested. We employed thapsigargin that exhibits increased inhibitory effects on SERCA compared to other inhibitors 31 . Our data showed that inhibition of SERCA significantly reduced lymphatic contractile frequency, amplitude and thus, diminished fractional pump flow and ejection fraction ( Fig. 2 and Table 2). These results imply that SERCA is an important mediator for lymphatic pumping by regulating phasic contractile activity, not by regulating lymphatic tonic contraction.
Diminished SERCA activity in metabolic diseases has been well established in various tissues, including cardiac, skeletal and smooth muscles, and in liver tissue. Consistent with our previous findings 9,10 , lymphatic contractility indexes, frequency of contraction and lymphatic fractional pump flow, were significantly reduced in MetSyn rats when compared to control group (Fig. 3C,E). Our data showing that the SERCA inhibitor, thapsigargin significantly reduces the frequency and amplitude of contractions of lymphatic vessels from control group, but not the vessels from MetSyn animals (Fig. 3C,D), suggesting that SERCA activity is already diminished in MetSyn lymphatic vessels. Ejection fraction was significantly lowered in the presence of thapsigargin compared to each cohort groups ( Table 2) and this might be due to the similar level of contractile amplitude between control and MetSyn.
Three distinct genes encoding SERCA 1, 2, and 3 produce more than 10 isoforms that are expressed in various muscles and non-muscle cells 52 . SERCA1a and b are predominant isoforms in skeletal muscle and SERCA3s are expressed in various non-muscle tissues. SERCA2a is predominantly expressed in both skeletal muscle type 1 Figure 4. Decreased SERCA2a expression coupled with elevation of MLC 20 phosphorylation in lymphatic vessels from MetSyn animals. (A) Representative images of SERCA2a and α-SMA staining for lymphatic vessels from control and MetSyn rats. Images were obtained with × 40 objective (NA = 0.9) on a confocal microscope. Scale bars, 50 μm. (B) Average projections were quantified and plotted for control (n = 18 fields of views from 6 vessels from 3 animals) and MetSyn (n = 30 fields of views from 6 vessels from 3 animals). Quantification of SERCA2a was plotted. # indicates p < 0.001 vs control. (C) Representative images of SERCA2b and α-SMA staining for lymphatic vessels from control and MetSyn rats. Images were obtained with × 40 objective (NA = 0.9) on a confocal microscope, and average projections were presented. Scale bars, 50 μm. (D) Images were quantified and plotted for control (n = 9 fields of views from 4 vessels from 2 animals) and (n = 10 fields of view from 4 vessels from 2 animals). (E) Representative images of MLC 20 phosphorylation staining for lymphatic vessels from control and MetSyn rats. Images were obtained with × 40 objective (NA = 0.9) on a confocal microscope. Scale bars, 50 μm. (F) Average projections were quantified and plotted for control (n = 17 fields of view from 6 vessels from 3 animals) and MetSyn (n = 12 fields of view from 6 vessels from 3 animals). # p < 0.008 versus control. Data are presented as mean ± SE. www.nature.com/scientificreports/ fiber and in adult cardiac muscle; while, SERCA2b is considered one of the major isoforms in smooth muscle cells 52,53 . Here, we report that lymphatic muscle cells express both SERCA2a and SERCA2b isoforms (Figs. 4A-D, 5D-G), further supporting our previous finding that lymphatic muscle presents a unique combination of muscle cell types that express both cardiac and smooth muscle contractile and regulatory proteins 12,[17][18][19] .
Previous studies in different metabolic disease models have shown a decreased SERCA activity in cardiac and vascular tissues, yet there were inconsistent results whether diminished SERCA activity resulted from decreased levels of SERCA expression or were independent of SERCA levels. Decreased SERCA2a protein expression was found in cardiac muscle coupled with impaired cardiac contractility in diabetic cardiomyopathy and in db/db mice 28,29,54 . Additionally, SERCA2a protein levels decreased in vascular smooth muscle cells from Type 1 and Type 2 diabetes animal models 27,55 . In contrast, studies have reported that there were conformational changes, altered SERCA regulatory molecules, or diminished SERCA activity without decreased SERCA expression in the cardiac tissues from db/db mice and in an insulin resistant rat model 56,57 . We found that SERCA2a expression was diminished significantly in MetSyn lymphatics whereas SERCA2b expression was similar between control and MetSyn groups (Fig. 4A-D). Thus, the decrease response to thapsigargin in MetSyn lymphatics that we observed could be due to the decrease levels of SERCA2a in lymphatic vessel.
One critical question we have not addressed is that how loss of expression/activity of SERCA result in a decreased lymphatic contractile frequency in MetSyn animal. It is well established in cardiac and smooth muscle literatures that intracellular calcium homeostasis that is primarily regulated by SERCA, ryanodine receptor, L-type Ca channel, Na + /Ca 2+ exchanger, and T-type calcium channel, plays an imperative role in regulating cardiac and smooth muscle contractile dynamics 58,59 . However, only limited studies on mechanisms of calcium regulation are available in lymphatic muscle. Lee & von der Weid have shown that T type Ca 2+ channel mediates chronotropy; whereas L-type Ca 2+ channel controls inotropic contraction of lymphatic vessel 24 . Additionally, while L-type calcium channel is partially responsible for differential contractile activity of mouse lymphatic vessels from various regions of the body, it controls both frequency and strength of phasic contractions in lymphatic vessel 15,60 . In the presence of ryanodine that inhibits ryanodine receptor, lymphatic vessels showed a decrease in contractile frequency and amplitude, without a change in tonic contraction 61 , suggesting critical roles of endoplasmic reticulum stored calcium in phasic contraction. Our data show that inhibition of SERCA by thapsigargin in lymphatic vessels diminished both phasic contractile frequency and amplitude without affecting the tone. Additionally, a decrease in SERCA expression and/or activity in MetSyn lymphatics resulted in diminished phasic contraction frequency and a tendency for reduced contraction amplitude with no change in lymphatic tone. Primary role of SERCA pump is reuptake of intracellular calcium back to sarcoplasmic reticulum, which is essential for muscle relaxation. Notably, lymphatic vessel relaxation is critical for allowing filling the lymph, consequently for efficient lymphatic pumping activity 62 . Hence, a decrease in SERCA expression/ activity either by thapsigargin or by MetSyn condition diminished lymphatic muscle phasic contraction. In addition, it also resulted in an increase in intracellular calcium, which induced Ca 2+ -dependent myosin light chain 20 phosphorylation 17,18 . Increase in intracellular calcium levels also regulate voltage-gated sodium and potassium channels 63 . Taken together, we propose that increase in intracellular calcium in lymphatic muscle could disrupt the homeostasis of other ions, such as K + , Cl − or Na + , by modulating respective ion channels in lymphatic muscle and consequently, it would change membrane potential and contractile frequency. Further studies are warranted how disruption in calcium homeostasis in lymphatic muscle influences other ion channels and thereby modulates phasic contractile frequency.
Decrease SERCA pump activity in lymphatic muscle would increase the cytosolic Ca 2+ in LMCs. Our cell culture data support this notion; results showed that in the basal condition, intracellular Ca 2+ levels were elevated in insulin resistant LMCs. Additionally, amplitude between peak and basal calcium levels was significantly decreased in insulin resistant LMCs, indicating decreased SERCA activity in LMCs treated with glucose and insulin. Increase in intracellular Ca 2+ will promote MLC 20 phosphorylation via Ca 2+ /calmodulin-MLCK pathway 22 . Increased phosphorylation of MLC 20 levels we observed in lymphatic vessels from MetSyn rats (Fig. 4E,F) corroborate with our previous finding that insulin resistant condition induced MLC 20 phosphorylation in LMCs 21 . Thus, we propose that increased intracellular Ca 2+ due to decreased SERCA2 expression and activity in LMCs (A) An example tracing shows that the SERCA inhibitor, thapsigargin increased intracellular Ca 2+ levels in the absence or presence of high glucose and insulin (n = 100 cells from 5 different cultured LMC dishes/groups). (B) Basal calcium level, before thapsigargin, was quantified and plotted. # indicates p < 0.001 versus control. $ indicates p < 0.001 versus control + insulin. * indicates p < 0.001 versus HG. (C) Amplitude between peak intracellular Ca 2+ after thapsigargin and basal was quantified for determining SERCA activity and plotted. # indicates p < 0.001 versus control. $ indicates p < 0.001 versus control + insulin. * indicates p < 0.001 versus HG. (D). Representative immunofluorescence images of lymphatic muscle cells stained with antibodies specific for the ER (SEC61, green), SERCA2a (red), and DAPI (blue). Images were obtained using × 40 objective (NA = 1.3) on a fluorescence microscope (n = 9 field of views from 3 cultured dishes/group). Scale bars, 50 μm. (E) SERCA2a relative fluorescence intensity was quantified and plotted. # indicates p < 0.032 versus control. Representative Western blots of SERCA2a (F) and SERCA2b (G) relative levels in LMCs treated with HG, insulin, or both together for 48 h. The relative expression of SERCA2a/β-actin and SERCA2b/βactin were quantified and plotted (n = 3/group). # indicates p < 0.05 versus control. $ indicates p < 0.05 versus control + insulin. (H) Representative Western blots show increased free extracellular calcium concentration induced MLC 20 phosphorylation in LMCs. The relative expression of pMLC 20 /total MLC 20 (n = 4/group). ^ indicates p < 0.1 compared to Ca 2+ free control. # indicates p < 0.05 versus Ca 2+ free control. Data are presented as mean ± SE.  www.nature.com/scientificreports/ Increasing SERCA expression or activity has been shown to provide protective effects on cardiac muscle and vascular smooth muscle cells in several metabolic diseases 26,29,54,55 . A small molecule, CDN1163, activates SERCA by allosteric mechanism and improves Ca 2+ homeostasis both in vivo and in vitro [35][36][37] . Our data show that SERCA activation significantly improves the lymphatic contractile frequency in MetSyn lymphatic vessels similar to control lymphatic vessels (Fig. 6F-H). Though the fractional pump flow was not significantly improved by SERCA activator in MetSyn lymphatic vessels, fractional pump flow was not significantly lower than control lymphatic vessels, suggesting partial improvement of contractile activity of MetSyn lymphatic vessels by SERCA activator. This partial improvement by SERCA activation in MetSyn group might be due to reduced SERCA2a expression in MetSyn lymphatic muscle.
In summary, the current study demonstrates that SERCA2 activation plays an important role in modulating lymphatic contractile function by regulating chronotropic and inotropic effects. The rat mesenteric lymphatic muscle expresses both SERCA2a and SERCA2b isoforms. MetSyn conditions decreased the levels of SERCA2a expression and impaired Ca 2+ regulation in LMCs that are coupled with increased MLC 20 phosphorylation. The impaired lymphatic pumping activity in MetSyn is due to diminished SERCA activity and activating SERCA partially improves contractile activity of lymphatic vessels from MetSyn rats. Therefore, it is possible that SERCA2 agonist could be used as a therapeutic strategy in enhancing lymphatic function in MetSyn or other metabolic diseases. Figure 7. Schematic representation of lymphatic vessel contraction under normal and MetSyn/insulin resistant conditions. In normal physiological condition, Ca 2+ homeostasis is critical for lymphatic pump regulation. The inhibition of SERCA directly diminishes lymphatic contractile activity and it is resulted in increasing intracellular Ca 2+ . Elevated intercellular Ca 2+ induce MLC 20 phosphorylation, a key contractile regulatory molecule that also influences lymphatic contractions. Insulin resistance in LMCs or high fructose diet-induced MetSyn condition in rats decreases SERCA2a expression and SERCA activity, disturbing intracellular Ca 2+ homeostasis and impairs lymphatic phasic contraction. Elevated intracellular Ca 2+ promotes MLC 20 phosphorylation via Ca 2+ /calmodulin and MLCK pathway 82 in insulin resistant LMCs. Normal insulin signaling in LMCs mediates its bioenergetics via proper glucose uptake and glycolysis and mitochondrial function 21 . Disrupted insulin signaling in insulin-resistant LMCs inhibits glucose uptake, induces mitochondrial dysfunction, and decreases bioenergetics 21 . Imbalanced cellular bioenergetics status causes energy stress or endoplasmic reticulum stress that may decrease SERCA expression or activity in insulin resistant LMCs and in MetSyn animals, which is not yet tested. Animal handling. Fifty-two male Sprague-Dawley rat weighing 150-180 g were ordered from Charles River for induction of the MetSyn. Twenty rats were given a high fructose diet (60% fructose, ID89247 Harlan Teklad, Envigo, Indianapolis, IN, USA), for 7-10 weeks to induce the MetSyn, while the remaining rats were given standard rodent chow. Water and each respective feed were available ad libitum. Rats from the control group were utilized for testing the effects of different dosage of thapsigargin, DMSO, and CDN1163, a SERCA activator (n = 12). Rats from the control and MetSyn group were used for isobaric functional analysis of mesenteric lymphatic vessel contractility (n = 14/group). The remaining 6 rats were used for IHC and RNA analysis. All animals were housed in a facility with 12-h light-dark diurnal cycle, accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care and maintained in accordance with the policies defined by the Public Health Service Policy for the Humane Care and Use of Laboratory Animals. All protocols had been approved by the University Laboratory Animal Compliance Committee at Texas A&M University prior to the commencement of the study.
Determination of metabolic parameters. High fructose diet generates a rat model of MetSyn as characterized by high insulin, glucose, triglycerides, and glucose levels along with impaired insulin sensitivity 38,[68][69][70] .
We also confirmed the development of MetSyn conditions by measuring plasma insulin, triglyceride, inflammatory cytokines in mesentery and cecum histopathological changes 9,10 . To confirm the pathology of high fructose diet-induced MetSyn we measured blood glucose using a glucometer and test strips before surgery. Heart was isolated, weighted, and then the left ventricle was dissected for measuring cardiac hypertrophy. Quadriceps, tibial anterior (TA), and soleus muscles were isolated and weighed for assessing sarcopenic obesity, which is systemic pathology due to metabolic disorders. Inguinal subcutaneous adipose tissue (SWAT) and epididymal white adipose tissue (EWAT) were dissected to determine the percent of subcutaneous and visceral fat.
Lymphatic vessel isolation and functional analysis. Rat mesenteric collecting lymphatic vessel isolation and cannulation were performed to test isobaric lymphatic pumping activity as described in our previous studies 9, 10,18 . Rats were anesthetized with Innovar-Vet (0.3 ml/kg), which is a combination of a Droperidol-fentanyl solution (Droperidol 20 mg/ml, fentanyl 0.4 mg/ml), and diazepam (2.5 mg/kg) intramuscularly. A midline excision was made and a loop of ileum was carefully exteriorized. Lymphatic vessels were carefully dissociated from the surrounding adipose tissue to prevent excess bleeding and use the following three criteria for defining lymphatic vessels: LMc culture and treatments. Primary rat mesenteric LMCs were obtained from mesenteric tissue explants of male Sprague-Dawley rats, as we have described in previous publications 12,21 . Briefly, 2-4 mesenteric lymphatic vessels were isolated under aseptic conditions from Sprague Dawley rats within a laminar hood. The vessels were cut into small segments (2-3 mm) and each small segment was transferred to a 35 mm tissue culture dish containing high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% antibiotic/antimycotic solution (penicillin/streptomycin). The vessel segment was attached by gently pressing the two ends of the vessel onto the plastic dish surface with forceps. The culture dish containing the vessel Scientific RepoRtS | (2020) 10:12320 | https://doi.org/10.1038/s41598-020-69196-4 www.nature.com/scientificreports/ was then carefully placed into an incubator maintained at 37 °C and 10% CO 2 . Lymphatic muscle cells, LMCs (~ 50 cells) migrated out from the vessel in 3-7 days, after which the lymphatic vessel-segment was removed from the petridish in the aseptic condition. At this condition, lymphatic endothall cells are not able to migrate and survive, whereas LMCs will sprout out from the vessel. We then allow the sprouted LMCs further to proliferate and once the plate becomes confluent, the cells were then trypsinized and designated as passage 1 (p1) and either split into more plates and maintained in the incubator or frozen and cryo-preserved. We have previously shown that LMCs comprise of a unique combination of cardiac and smooth muscle components 12 . We routinely analyze each batch of LMCs at stages p2-p6 for expression of both cardiac and smooth muscle phenotype, using cardiac troponin C, cardiac β myosin heavy chain, alpha smooth muscle actin, SM22, and caldesmon antibodies. Phenotypes of LMCs were also verified using qRT-PCR with α/β-myosin heavy chain, cardiac troponin, and α-smooth muscle actin as we have previously described 12 . Cells were not used beyond passage 6. LMCs were cultured in DMEM, containing 10% FBS, and 1% triple antibiotics and maintained at 37 °C in 10% CO 2 incubator. LMCs were plated in 24-well culture plates and then grown to ~ 70-80% confluence. The cells were serum starved for 24 h and treated with 5 mM glucose (control) or high glucose (HG; 25 mM), with or without insulin (100 nM) for 48 h as we reported in a previous study 21 . To test the effects of free intracellular Ca 2+ , different concentrations of pCa solutions (between pCa 7.5 and 3.5) were prepared based on Ca free DMEM using a software, MaxChelator as described 74,75 . After serum starvation, LMCs were treated with Ca 2+ free DMEM and different concentrations of free calcium (pCa 7.5-3.5) for 30 min. Proteins were isolated as described in our previous studies 21,46 . intracellular ca 2+ measurement in LMcs. Intracellular Ca 2+ levels were measured using fura-2AM in phenol-free DMEM media as described [76][77][78] . LMCs were plated in glass bottom chamber and treated when LMCs reached 60% confluence. Cells were loaded with Fura-2AM (2 μM) in the dark for 30 min at 37 °C. Cells were then washed with phenol-free DMEM and incubated for another 30 min for de-esterification. Pairs of fluorescent images were taken by exposure to 340-and 380 nm double excitation with interference filters at selected wave length (Lamda DG-5, Sutter Instruments, Novato, CA, USA) and 510 nm emission wavelengths using an epifluorescence microscopy system (Nikon Eclipse Ti, Nikon, Melville, NY USA). The individual traces of fura-2AM dye from multiple LMC region of interest, ROI were simultaneously measured from a field of view (~ 20 ROIs/plate). The fura-2 fluorescence ratio was collected for each cell throughout the experiments (NIS-Elements software, Nikon, Melville, NY, USA). Background fluorescence was determined before the start of the experiment. Background fluorescence was subtracted and fluorescence ratios of images at 340 and 380 nm were determined. Calcium levels were measured using fura-2AM calcium imaging calibration kit (Thermo Fisher) where it had zero to 10.0 mM CaEGTA (1.35 μM free calcium buffer) prior and post experiments. The interrelationship of the free Ca 2+ concentration and the ratio was calculated according to manufacturer's guideline 79 . Basal Ca 2+ levels were measured in phenol free-DMEM. Voltage-gated calcium channel were activated by depolarization with high (80 mM) K + solution. SERCA activity was assessed with thapsigargin (5 μm).

Immunofluorescence analyses.
To determine the relative levels of SERCA2a, SERCA2b, and MLC 20 phosphorylation, rat mesenteric lymphatic vessels were prepared for immunofluorescence as indicated in previous studies 18,46 . Multiple adjacent lymphatic vessels were isolated and fixed in 2% paraformaldehyde for 60 min at room temperature. After fixation, vessels were washed in PBS and permeabilized for 5 min in − 20 °C methanol. Vessels were then blocked in 5% goat serum (with 1% BSA) for 1 h, subsequently cut in half with one part being used for the experimental treatment and the other part serving as the negative control. Vessels were incubated with SERCA2a (A010-23, Badrilla, Leads, UK), SERCA2b (A010-24, Badrilla, Leads, UK), α-Smooth muscle actin (A2547, Sigma-Aldrich, St. Louis, MO, US), or phospho MLC 20 (3,645, Cell signaling, Beverly MA, US) antibody in blocking solution overnight. Vessels were then washed and incubated with host matched secondary antibody (1:200) conjugated with Alexa Fluor 488 or Alexa Fluor 594 after washed three times. Vessels were then washed three times and mounted using ProLong gold antifade mounting media. Vessels were scanned in 0.5μm z-axis steps using PLAPLSM 40 × objective (NA = 0.9), Olympus IX-71 inverted microscope with Fluoview 300 confocal scanning. Average projections of series sections were reconstructed and quantified using Image J software 80 . Negative control vessel segments were subjected to the same procedures as the experimental treatments except that negative controls were incubated with corresponding host IgG instead of the primary antibody. Negative controls were scanned at the same microscope settings as the experimental treatments to allow for valid comparison of the relative fluorescent intensities. WGA staining. Membrane specific dye, wheat germ agglutinin (WGA), was used to test MetSyn induced myopathies as described elsewhere 81 . Frozen OCT block of TA, soleus, and left ventricles were cut 10 micron thickness using cooled cryostat (Leica CM1850, Leica Biosystems Inc., Buffalo Grove, IL, USA) at − 20 °C. Sections were fixed in cooled acetone (− 20 °C) for an hour. The slides were then washed three times in PBS for 5 min and incubated with WGA for 10 min at room temperature. Sections were washed and mounted using ProLong gold antifade mounting media. Images were taken with Olympus BX41 fluorescence microscope using UPlanApo × 10 (NA = 0.4) or UplanApo X20 objective (NA = 0.7) (Olympus America, Melville, NY, USA) at an excitation peak of 545 nm with an emission spectral peak of 610 nM.

ER and SERCA2a staining in LMCs.
Immunofluorescence experiments in LMCs were performed as described earlier, with some modification 21 , to determine SERCA2a expression and localization on ER. Briefly, the treated LMCs were fixed with 2% paraformaldehyde and permeabilized with cooled methanol for 4 min at 4 °C. Cells were then blocked with 1% BSA with 5% goat serum for 1 h at room temperature. Cells incu-Scientific RepoRtS | (2020) 10:12320 | https://doi.org/10.1038/s41598-020-69196-4 www.nature.com/scientificreports/ bated SERCA2a antibody (1:100) in blocking buffer for two hours at room temperature. After three washes, cells were incubated with Alexa Fluor 594 (goat anti-rabbit, 1:200) for 1 h at room temperature in the dark. Since SEC61, specific to ER protein antibody is also from the rabbit, we used commercially pre-labeled SEC61 antibody (ab205794, Abcam, Cambridge, MA, USA), generated with an antibody labeling kit (A20181, Thermo Fisher, Waltham, MA, USA) and applied after SERCA2a antibody. Cell were counterstained for the nucleus and mounted using Prolong Gold antifade mounting medium with DAPI. Images were taken with Olympus BX41 fluorescence microscope using UPlanFLN X40 objective (NA = 1.3) (Olympus America, Melville, NY, USA) and SERCA2a fluorescence intensity was measured with Image J.
protein isolation and Western blot analysis. Protein expression was quantified by Western blot analysis 21,46 . In brief, LMC protein lysates were prepared from LMCs and separated onto a 4-20% gradient SDSpolyacrylamide gel and transferred, and Western blot analysis was performed. SERCA2a, SERCA2b, MLC 20 phosphorylation, MLC 20 , and β-actin (1:1,000) were used. Membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. Protein detection was conducted with an enhanced chemiluminescence system and visualized via image processor Fuji LAS-4000 Mini (GE Healthcare Bio-science, Pittsburg, PA, USA). β-actin expression was used as the loading control. Densitometric analyses was carried out using Image J (National Institute of Health, NIH, Bethesda, MD, USA). For quantification, experiments were repeated three times for each sample, and the resulting means ± SE were calculated (n = 3/group).
Statistical analysis. Animal characteristics (e.g., bodyweight, glucose level, % fat, muscle weight, cross sectional area, etc.,) were analyzed by independent Student's t tests after performing Leven's test for equal variance. Different dose effects of thapsigargin and CDN1163 in lymphatic contractility parameters were analyzed using One-way ANOVA with Dunnett's post hoc test to find the treatment dose differences compared to control. Control and MetSyn lymphatic contractile parameters in the presence or absence of thapsigargin or CDN1163 were analyzed using Two-way ANOVA with Bonferroni's post hoc test to examine the interaction and main effects between two factors: diet and treatment (i.e., thapsigargin,CDN1163) . When we found a main effect or interaction, we performed follow-up ANOVA using Fischer's LSD post hoc test to find between (i.e., diet) or within group (i.e., thapsigargin/CDN1163). Levene's statistics also performed to test the homogeneity of variances. LMC data including Ca 2+ were analyzed using Two-way ANOVA with Bonferroni's post hoc test (glucose and insulin) as described earlier. Once we find any interaction or main effects, we performed follow-up ANOVA using Fisher's LSD test. The homogeneity of variance was performed using Levene's statistics. Statistical analyses were performed using SPSS software (IBM Corp, Armonk, New York, USA). All values are expressed as mean ± SE. The significance level was set at p < 0.05. All graphs were generated with Prism 5 (GraphPad Software, La Jolla, CA, USA).

Data availability
The datasets analyzed during the current study are available from the corresponding author on reasonable request.