LPL/AQP7/GPD2 promotes glycerol metabolism under hypoxia and prevents cardiac dysfunction during ischemia.

Fatty acid constitutes a major energy substrate in the heart to fuel contraction under aerobic conditions. Ischemia downregulates fatty acid metabolism to adapt to the limited oxygen supply and makes glucose the preferred substrate. However, the mechanism of the myocardial metabolic shift during ischemia remains unknown. Here, we show that cardiomyocyte secretion of lipoprotein lipase (LPL), a principal enzyme that converts triglycerides to free fatty acids and glycerol, increased during myocardial infarction (MI). Cardiomyocyte-specic LPL de�ciency enhanced cardiac dysfunction and apoptosis following MI. De�ciency of aquaporin 7 (AQP7), a glycerol channel in cardiomyocytes, increased the myocardial infarct size and apoptosis in response to ischemia. Ischemic conditions activated glycerol-3-phosphate dehydrogenase 2 (GPD2), which converts glycerol-3-phosphate into dihydroxyacetone phosphate to facilitate ATP synthesis from glycerol. Conversely, GPD2 de�ciency exacerbated cardiac dysfunction after acute MI. Together, these results identify that LPL/AQP7/GPD2-mediated glycerol metabolism plays an important role to bridge glucose and lipid metabolism in MI and prevent myocardial ischemia-related damage.


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Fatty acid constitutes a major energy substrate in the heart to fuel contraction under aerobic conditions 1 .
Ischemia downregulates fatty acid metabolism to adapt to the limited oxygen supply and makes glucose the preferred substrate 2,3 .However, the mechanism of the myocardial metabolic shift during ischemia remains unknown.Here, we show that cardiomyocyte secretion of lipoprotein lipase (LPL), a principal enzyme that converts triglycerides to free fatty acids and glycerol 4 , increased during myocardial infarction (MI).Cardiomyocyte-speci c LPL de ciency enhanced cardiac dysfunction and apoptosis following MI.De ciency of aquaporin 7 (AQP7), a glycerol channel in cardiomyocytes 5,6 , increased the myocardial infarct size and apoptosis in response to ischemia.Ischemic conditions activated glycerol-3phosphate dehydrogenase 2 (GPD2), which converts glycerol-3-phosphate into dihydroxyacetone phosphate to facilitate ATP synthesis from glycerol 7 .Conversely, GPD2 de ciency exacerbated cardiac dysfunction after acute MI.Together, these results identify that LPL/AQP7/GPD2-mediated glycerol metabolism plays an important role to bridge glucose and lipid metabolism in MI and prevent myocardial ischemia-related damage.
The heart has a high rate of ATP demand to sustain contractile activity and maintain tissue perfusion.
Approximately 60-90% of cardiac ATP is produced by the oxidation of fatty acids, whereas the remaining 10-40% comes from the oxidation of glucose, lactate, ketone bodies and amino acids 1 .Thus, although fatty acids constitute the predominant substrate used in the heart, the cardiac metabolic network is highly exible with regard to use of other substrates depending on physiological and pathological stress such as exercise, pregnancy, myocardial infarction (MI), and heart failure 2 .The reciprocal relationship between fatty acid and glucose metabolism was rst described in the 1960s 8 .Speci cally, the increased generation of acetyl CoA derived from fatty acid oxidation decreases glucose oxidation in the heart through inhibition of pyruvate dehydrogenase, the enzyme that catalyzes pyruvate decarboxylation, a key irreversible step in glucose oxidation 9 .Conversely, increased acetyl CoA generation from glucose oxidation inhibits fatty acid oxidation by inhibiting carnitine palmitoyltransferase 1, which enhances fatty acid transport into the mitochondria.MI, which is de ned as myocardial cell death owing to prolonged ischemia, remains the leading cause of mortality worldwide 10 .The onset of myocardial ischemia results from an imbalance between oxygen supply and demand.It is generally accepted that under hypoxic conditions, cardiac metabolism shifts from fatty acids to glucose 1 , which is more e cient with respect to ATP production per O 2 consumed 3 .
Accumulating evidence suggests that modulating cardiac energy metabolism by increasing glucose oxidation and decreasing fatty acid oxidation, can improve cardiac function in heart disease 11 ; however, various factors increase the concentration of plasma free fatty acids (FFA), such as hormonal state in response to myocardial ischemia 8 .This contradiction between the detrimental effects of fatty acid oxidation and increased plasm FFA levels in MI suggests that lipoprotein lipase (LPL), which is the principal enzyme that converts triglycerides in the circulation to FFA 4 , mediates other metabolic pathways in MI.LPL is produced from cardiomyocytes, skeletal muscle and adipose tissue to control local fatty acid uptake 12 , and a genetic study indicates that LPL activation reduces coronary artery disease risk 13 .Glycerol is generated in the process of LPL-catalyzed breakdown of the triglyceride component of lipoproteins to provide fatty acids 14 .Aquaporin 7 (AQP7) is an aquaglyceroporin that facilitates the transport of glycerol across cell membranes into the heart 5,6 .AQP7 de ciency reduces glycerol uptake in the heart and exacerbates pressure overload-induced heart failure 15 , but the role of glycerol as a substrate for energy production in cardiomyocytes under hypoxia conditions remains unclear.
To investigate whether acute MI increases cardiac LPL expression, the left coronary artery in wild-type (WT) mice was ligated.LPL was assayed by immunostaining of sections using an antibody against LPL 1 h after the ligation (Fig. 1A and 1B).MI increased the intensity of LPL at the capillary endothelium in the infarct area, suggesting that the ischemic conditions enhance LPL expression in vivo.To investigate whether hypoxia conditions affect LPL secretion from isolated adult murine cardiomyocytes, LPL on these cells under 1-h hypoxia conditions was ascertained by immunostaining (Fig. 1C and 1D).Hypoxia conditions signi cantly increased LPL intensity on the cardiomyocyte surface, suggesting that hypoxia enhanced cardiac LPL secretion.To examine the functional signi cance of cardiac LPL under ischemic conditions in vivo, we used mice with tamoxifen-inducible cardiomyocyte-speci c de ciency for LPL (cmc-LPL KO).Immunostaining analysis revealed decreased LPL in the hearts of cmc-LPL KO mice (Fig. 1E).No differences were observed in hepatic LPL expression between cmc-LPL WT and cmc-LPL KO mice (Figure S1).Notably, cardiomyocyte-speci c LPL de ciency suppressed MI-induced LPL expression at the capillary endothelium in the heart, suggesting that LPL is synthesized in cardiomyocytes under ischemic conditions.We performed echocardiography to assess cardiac functionality 1 day after the left coronary artery ligation (Fig. 1F).In control mice, 1-day coronary artery ligation resulted in signi cantly decreased cardiac function, with cardiac LPL de ciency exacerbating the MI-induced cardiac dysfunction.
To examine whether cardiac LPL de ciency affects the cardiac apoptosis induced by the ligation, apoptotic cardiomyocytes were assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining of heart sections 1 day after the ligation (Fig. 1G).LPL de ciency increased MIinduced apoptosis.
As glycerol is produced when LPL converts triglycerides to FFA 14 , we next investigated whether glycerol is a substrate for energy production in cardiomyocytes under hypoxia conditions.Isolated adult murine cardiomyocytes were incubated with glycerol and exposed to hypoxia conditions (Fig. 2A).After 4-h exposure to normoxia or hypoxia, we identi ed damaged cardiomyocytes using trypan blue vital staining.
Glycerol treatment increased cardiomyocyte cell viability already under normoxia conditions in a dosedependent manner; however this effect was enhanced under hypoxia conditions.AQP7 is expressed in adipose tissue, skeletal muscle and the heart, and serves as a glycerol channel 15 .To investigate whether the protective effect of glycerol under cardiac hypoxia requires AQP7, cardiomyocytes were isolated from AQP7 +/+ and AQP7 −/− mice.AQP7 de ciency decreased glycerol-increased cardiomyocyte cell viability (Fig. 2B).To examine whether AQP7 de ciency increased MI-induced cardiac apoptosis, apoptotic cardiomyocytes were identi ed by TUNEL staining of heart sections 1 day after the left coronary artery ligation (Fig. 2C and 2D).No difference in apoptosis was observed between AQP7 +/+ and AQP7 −/− in control animals whereas AQP7 de ciency signi cantly increased MI-induced apoptosis.To determine whether glycerol attenuates the progression of MI-induced cardiac dysfunction in mice, infarct changes were ascertained in PicroSirius red-stained Sect.7 days following the ligation (Fig. 2E and 2F).AQP7 de ciency increased the infarct area in the heart.We also performed echocardiography to assess cardiac functionality (Fig. 2G and 2H).In control mice, 7-day MI resulted in a signi cantly decreased fractional shortening, with the response enhanced in AQP7 −/− mice.In addition, atrial natriuretic peptide (ANP) in the heart, which is increased upon cardiac dysfunction, was elevated in AQP7 −/− mice 7 days following ligation (Fig. 2I).
After the cells capture glycerol, glycerol kinase (GK) catalyzes the phosphorylation of glycerol to yield G3P 16 .GK expression is considered to be restricted to the liver, kidney, and skeletal muscle.To examine Gk gene expression in the heart, we performed quantitative polymerase chain reaction (qPCR) analysis of various murine tissues (Fig. 3A), which revealed Gk was also expressed in the heart.In turn, glycerol-3phosphate dehydrogenase 2 (GPD2), which is anchored to the mitochondrial membrane, oxidizes glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP) in the cytoplasm 7 .To examine GPD2 enzymatic activity on cardiac mitochondria, mitochondria were isolated from the heart; these exhibited GPD2 activity in vitro (Fig. 3B).Next, as Ca 2+ increases GPD2 activity on mitochondria in the liver but not in the brain of rat 17 , cardiac mitochondria were incubated with high concentration of Ca 2+ .This increased GPD2 activity whereas treatment with EDTA suppressed Ca 2+ -induced GPD2 activation, suggesting that the high Ca 2+ concentration enhances GPD2 activity in the heart.To examine whether MI affects GPD2 enzymatic activity in the heart, mitochondria were isolated from the heart 1 h after sham surgery or coronary artery ligation (Fig. 3C).Ischemic conditions signi cantly increased the cardiac GPD2 enzymatic activity.Next, we examined whether glycerol is involved in the energy metabolism in cardiomyocytes.As 4-h hypoxia was shown to decrease cardiomyocyte cell viability (Fig. 2A), we examined intracellular ATP concentrations in isolated adult murine cardiomyocytes under 1-h hypoxia conditions to avoid cell viability effects on ATP production (Fig. 3D).Notably, 1-h hypoxia conditions did not affect cell viability and no difference between control and glycerol treatment was observed.
Conversely, 1-h hypoxia signi cantly decreased ATP production in cardiomyocytes (Fig. 3E).Although glycerol treatment did not increase ATP production under normoxia conditions, glycerol-mediated ATP production was signi cantly increased under hypoxia conditions.In comparison, the glycerol-dependent ATP production under early hypoxia conditions was suppressed by the GPD2 inhibitor in a dosedependent manner, whereas signi cant effects on ATP production under normoxia conditions were not detected.To evaluate the role of GPD2 in vivo, we performed coronary artery ligation in GPD2 +/+ and GPD2 −/− mice.In control mice, cardiac dysfunction was observed at 2 and 4 weeks after the ligation (Fig. 3F).GPD2 de ciency signi cantly exacerbated MI-induced cardiac dysfunction and increased infarct area in the heart (Fig. 3G).
Here, we demonstrated that glycerol is a substrate for energy production in cardiomyocytes under hypoxia conditions (Fig. 3H).Glycerol plays biochemically important roles by serving as the backbone of glyceride lipids 18 .Once glycerol enters the major pathways of carbohydrate metabolism such as glucose metabolism, it also functions as an energy substrate.For example, intracellular G3P and pyruvate are synthesized upon culture of neonatal rat cardiomyocytes with glycerol 19 .In vivo, the use of 14 C-labeled glycerol reveals that an increase of heart rate in rat induces a concomitant increase in glycerol uptake in the heart 20 .
In particular, the use of glycerol as a fuel is mediated via AQP7 in the heart under pathological situations such as MI and heart failure.The expression of AQP7, which is the most prominent aquaglyceroporin in the heart, increases under conditions of altered energy supply such as diabetes mellitus, fasting, exercise, and high-protein diets 6 .Immunohistochemical staining revealed that AQP7 is localized in capillaries of the heart 21 , whereas in vivo experiments using AQP7 −/− mice indicated that AQP7 acts as a glycerol channel in cardiomyocytes.Speci cally, although AQP7 −/− mice exhibit normal cardiac histology and morphology under basal conditions, AQP7 de ciency exacerbates pressure overload-induced cardiac hypertrophy and isoproterenol-induced cardiac dysfunction and increases mortality following pressure overload-induced heart failure 15,22 .
The heart avidly acquires lipids both from circulating FFA and esteri ed fatty acids bound to lipoproteins 23 , with esteri ed FFA constituting a major source of cardiac lipids whereas circulating FFA is of minor importance as fuel for the heart.Because the water solubility of FFA is limited, they must undergo esteri cation with glycerol to form triglycerides, which make up a signi cant portion of lipoprotein triglycerides in the circulation 24 .LPL is synthesized in parenchymal cells such as adipocytes and cardiomyocytes.GPI-anchored LPL transgenic mice, in which LPL is anchored to cardiomyocytes, are unable to transport LPL from cardiomyocytes to the vascular lumen, suggesting that LPL is produced in cardiomyocytes and is transferred to the apical side of endothelial cells, where the enzyme functions in the heart 25 .Moreover, tamoxifen-inducible cardiac LPL de cient mice (MerCreMer LPL ox/ ox ) increase plasma triglyceride concentration and decrease left ventricular fractional shortening 4 weeks after tamoxifen treatment 26 .Thus, in the present study we ligated the coronary artery of cmc-LPL WT and cmc-LPL KO mice 5 days after tamoxifen treatment to avoid the basal cardiac dysfunction caused by LPL de ciency.Our in vivo investigation indicated that MI causes LPL secretion from cardiomyocytes, which in turn prevented MI-induced cardiac dysfunction.Alternatively, under ischemic conditions, fatty acid translocase translocates away from the sarcolemma in the heart to limit fatty acid uptake 27 .The contradiction between downregulation of fatty acid oxidation and increase of LPL secretion in the heart exposed to MI supports our model that the upregulation of glycerol metabolism by LPL, which hydrolyzes circulating triglycerides into FFA and glycerol in the vascular lumen, prevents cardiac dysfunction.
Although GPD2 plays an important role in physiological and pathological situations such as the release of insulin in pancreatic islets β cells and macrophage in ammatory responses 28,29 , the role of GPD2 in the heart remains unclear.Here, we found that GPD2 de ciency did not affect basal cardiac function and that GPD2 constitutes an important regulator bridging glucose and lipid metabolism in MI.In glycerol metabolism, ATP synthesis with conversion from DHAP to pyruvate, which is oxygen-independent, assists in ATP production under anaerobic conditions.These ndings indicated that the use of glycerol as energy source by GPD2-mediated myocardial metabolism is suitable under hypoxia conditions during MI.
Moreover, the correlation between LPL activity and phosphorylation of AMP-activated protein kinase (AMPK), which is an essential component of the adaptive response to cardiomyocyte stress during MI, is revealed by fasted rats 30 .The intracellular Ca 2+ concentration in cardiomyocytes increases under MI, additionally, Ca 2+ is a modulator of GPD2 isolated from rat liver in vitro 17 .We found that Ca 2+ activated GPD2 on mitochondria isolated from the heart and that coronary artery ligation increased GPD2 enzymatic activity in vivo.These ndings suggested that AMPK and Ca 2+ may mediate ATP production from glycerol in MI via LPL and GPD2 activation; however, the mechanism through which glycerol metabolism is activated under ischemia conditions requires further investigation.
In conclusion, our data revealed that MI-induced LPL secretion from cardiomyocytes increased to prevent myocardial ischemia.AQP7 acts as a glycerol channel in cardiomyocytes during MI.GDP2 causes intracellular glycerol to enter the major pathway of carbohydrate metabolism, glucose metabolism, to produce ATP.The ndings that LPL/AQP7/GPD2-mediated glycerol metabolism plays an important role to bridge glucose and lipid metabolism in MI makes this glycerol pathway a promising target for therapeutic intervention in acute MI.
Declarations AUTHOR CONTRIBUTIONS

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Figure 1 MI
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Figure 1 MI
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Figure 3 GPD2
Figure 3 S.I., T.Y. and S.Y. performed most of the in vitro and in vivo experiments, analyzed, and discussed data, and commented on the manuscript.Y.M., S.E., T.S., T.T., K.K. and Y.S. performed in vivo experiments and mouse generation.H.Y. generated LPL ox mice.N.W. and S.O.discussed data and generated αMHC-Cre-ERT2 mice.N.K. and T.K. commented on the manuscript and generated GPD2 KO mice.Y.B., K.O., N.O., and T.M. supervised the study and commented on the manuscript.M.T. initiated the study, performed in vitro and in vivo experiments, analyzed and discussed data, and wrote the manuscript.