Matthieu Ruiz,1,2 Lise Coderre,1,2,3 Dominic Lachance,1,2 Valérie Houde,1,2

Cécile Martel,2 Julie Thompson Legault,1,2 Marc-Antoine Gillis,2 Bertrand Bouchard,1,2

Caroline Daneault,1,2 André C. Carpentier,4 Matthias Gaestel,5 Bruce G. Allen,2,3,6 andChristine Des Rosiers1,2,3

MK2 Deletion in Mice PreventsDiabetes-Induced Perturbations inLipid Metabolism and CardiacDysfunctionDiabetes 2016;65:381–392 | DOI: 10.2337/db15-0238

Heart disease remains a major complication of diabetes,and the identification of new therapeutic targets isessential. This study investigates the role of the proteinkinase MK2, a p38 mitogen-activated protein kinasedownstream target, in the development of diabetes-induced cardiomyopathy. Diabetes was induced incontrol (MK2+/+) and MK2-null (MK22/2) mice using re-peated injections of a low dose of streptozotocin (STZ).This protocol generated in MK2+/+ mice a model of di-abetes characterized by a 50% decrease in plasma in-sulin, hyperglycemia, and insulin resistance (IR), as wellas major contractile dysfunction, which was associatedwith alterations in proteins involved in calcium handling.While MK22/2-STZ mice remained hyperglycemic, theyshowed improved IR and none of the cardiac functionalor molecular alterations. Further analyses highlightedmarked lipid perturbations in MK2+/+-STZ mice, whichencompass increased 1) circulating levels of free fattyacid, ketone bodies, and long-chain acylcarnitinesand 2) cardiac triglyceride accumulation and ex vivopalmitate b-oxidation. MK22/2-STZ mice were also pro-tected against all these diabetes-induced lipid alter-ations. Our results demonstrate the benefits of MK2deletion on diabetes-induced cardiac molecular andlipid metabolic changes, as well as contractile dysfunc-tion. As a result, MK2 represents a new potential thera-peutic target to prevent diabetes-induced cardiacdysfunction.

Diabetes remains a worldwide health problem, which isassociated with a high rate of mortality primarily as aconsequence of cardiovascular diseases. Despite advancesin our understanding of the pathophysiology of diabetes,its prevalence is estimated to rise in the next decade.Current drug therapies aim at normalizing glucose levels,thereby preventing complications (1). Although thesetherapeutic agents efficiently lower blood glucose levels,there is a progressive loss of their efficacy in maintainingglycemic control over time (2). Hence, the development ofnew strategies for the treatment of diabetes remains es-sential and constitutes an active field of research.

In addition to glucose, diabetes is associated withperturbations in lipid metabolism in various tissues,including the heart (3). As a result, targeting lipid me-tabolism has been proposed as an interesting alternativestrategy to improve diabetes-induced cardiac dysfunc-tion. Consistent with this notion, pharmacologicalagents, such as troglitazone (4) or peroxisome proliferator–activated receptor a (PPARa) agonists, which modu-late fatty acid (FA) metabolism, have shown benefits inanimal models of cardiac dysfunction (5,6). Progress inthis area requires, however, a better understanding ofthe molecular mechanisms underlying these lipid alter-ations. In this regard, the current literature has involvedmitogen-activated protein kinases (MAPKs), such as ex-tracellular signal–related kinase 1/2, c-Jun N-terminal

1Department of Nutrition, Université de Montréal, Montréal, Québec, Canada2Research Center, Montreal Heart Institute, Montréal, Québec, Canada3Department of Medicine, Université de Montréal, Montréal, Québec, Canada4Department of Medicine, Centre de Recherche du Centre Hospitalier Universi-taire de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada5Institute of Biochemistry, Hannover Medical School, Hannover, Germany6Department of Biochemistry, Université de Montréal, Montréal, Québec, Canada

Corresponding authors: Christine Des Rosiers, christine.des.rosiers@umontreal.ca,and Bruce G. Allen, bruce.g.allen@umontreal.ca.

Received 20 February 2015 and accepted 5 November 2015.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0238/-/DC1.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 65, February 2016 381

METABOLISM

kinase, or p38MAPK in metabolic syndrome, obesity, anddiabetes (7).

Among these MAPKs, p38MAPK was described ashaving a key role in the pathophysiology of diabetes (8).p38MAPK modulates both lipid and glucose metabolism(8), mediates insulin resistance (IR) (9), and contributesto diabetes-induced cardiac dysfunction (10). Inactivationor inhibition of p38MAPK improves insulin signaling inthe liver (11) and alleviates the development of cardiacdysfunction in rodent models of diabetes (10). To the bestof our knowledge, however, none of the p38MAPK inhibi-tors have been approved for medical use in human subjectsbecause of numerous side effects, including hepatotoxicityor cardiotoxicity (12).

To circumvent p38MAPK inhibitor toxicity, an alter-native strategy is to target one of the downstream targetsof this kinase, such as PRAK/MK5, MSK1/2, MK3, orMK2. Interestingly, similar to p38MAPK, MK2 wasrecently reported to be activated in diabetic liver (11)and heart (13), and MK2 inhibition improves glucosehomeostasis and insulin sensitivity in obese mice (14).Furthermore, mice lacking both MK2 and MK3 displaydifferences in the expression of various genes involvedin lipid and carbohydrate metabolism in skeletal muscle(15). In addition, cardiomyocytes from MK2/MK3 double-knockout mice showed both increased contractility andsarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a(SERCA2a) expression (15). However, whereas MK2 deletionin mice has been reported to be beneficial in models ofcardiac ischemia-reperfusion and pressure overload–inducedhypertrophy (16,17), to the best of our knowledge, the in-volvement of MK2 in diabetes-induced metabolic alter-ations and cardiac dysfunction has not yet been examined.Thus, the objective of this study was to investigate whetherMK2 could impact on diabetes-induced complications usinga murine pan-MK2 knockout model, in comparison withtheir control littermates, in a model of diabetes induced byrepeated injections of a low dose of streptozotocin (STZ).Collectively, our results show that the absence of MK2prevented diabetes-induced systemic and cardiac lipid me-tabolism perturbations as well as cardiac dysfunction.

RESEARCH DESIGN AND METHODS

A protocol timeline is shown in Supplementary Fig. 1.

ChemicalsSources of chemicals, biological products, and 13C-labeledsubstrates as well as the albumin dialysis procedure (BSAfraction V; Intergen) have been described previously (18).

AnimalsAnimal experiments were approved by the Animal Re-search Ethics Committee of the Montreal Heart Institutein agreement with the guidelines of the Canadian Councilon Animal Care. The generation of MK2-null (MK22/2)mice has been described previously (19), whereas genotypingis described in the Supplementary Data. MK22/2 mice areviable and fertile and have no obvious physiological

defects. Mice were housed in a specific pathogen-free fa-cility under a 12-h light/dark cycle at constant tempera-ture and were provided food and water ad libitum.

Diabetes ModelFive-week-old male control (MK2+/+) and MK22/2

mice were fed a standard diet (3.02 kcal/g; RodentDiet 5001; LabDiet) and, after a 2-week adaptationperiod, received daily intraperitoneal STZ injections(40 mg/kg i.p.; Sigma-Aldrich) or vehicle (0.5 mol/L cit-rate buffer, pH 4.5). Mice were considered to be diabeticonce their fasted blood glucose levels had reached orexceeded a concentration of 12.5 mmol/L on 5 consecu-tive days, which required between five and eight injec-tions. Mice were observed for a total of 15 weeks. Bodyweight and food intake were measured weekly.

Insulin and Glucose Tolerance TestsIntraperitoneal insulin tolerance tests (ipITTs; insulin0.6 units/kg; Novolin ge) were performed at week 9 after a5-h fast. For oral glucose tolerance tests (OGTTs), performedat week 10 after a 16-h fast, glucose (dextrose solution2 g/kg; Hospira) was delivered into the stomach using a gavageneedle (20-gauge 3 1.5 inches, with 1.9-mm tip; CadenceScience). For both tests, blood was drawn from the saphe-nous vein, and the glucose level was measured prior to and15, 30, 45, 60, 90, and 120 min after insulin injection ordextrose gavage. Insulin levels during the OGTT were ex-amined at time 0, 15, 30, and 60 min. These tests wereassessed, and results were analyzed according to the pub-lished standard operating procedure (20).

In Vivo Cardiac Function: Millar CatheterizationFed mice were anesthetized with 2% isoflurane in 100%O2 at a flow rate of 1 L/min, and body temperature wasmonitored and maintained at 37°C using a heating pad.Hemodynamic parameters were measured using a micro-tip pressure transducer catheter (1.4F; Millar Instru-ments) inserted into the left ventricle through thecarotid artery as previously described (21). Data wereanalyzed using iox software version 2.5.1.6 (emka TECH-NOLOGIES, Falls Church, VA).

Blood and Plasma AnalysesBlood was collected at various time points. Plasma wasobtained after centrifugation at 1,200g for 10 min at 4°Cand stored at 280°C. Analyses performed after a 5-hfast included the following: 1) in blood (from saphenousvein), glycemia (Accu-Chek Aviva glucometer; Roche)and ketonemia (Blood b-Ketone test strips, PrecisionXtra; Abbott) and glycated hemoglobin (HbA1c) levels(Bayer A1C Now; Bayer) and 2) in plasma, insulin(Mouse Insulin ELISA; Albco). Other analyses performedin the fed state are as follows: 1) plasma free FAs (FFAs)(Fatty Acid Assay Kit; Biovision) and 2) plasmafree carnitine and 17 acylcarnitines (ACs) using an elec-trospray ionization tandem mass spectrometry method(22) with some modifications (for details, see Supple-mentary Data).

382 MK2 Deletion Improves Diabetic Cardiomyopathy Diabetes Volume 65, February 2016

Cardiac Tissue AnalysesLevels of transcripts (by quantitative RT-PCR), proteins(by immunoblotting), and triglycerides (TGs; assessed asdescribed previously [23] from their FAs analyzed by gaschromatography–mass spectrometry) were performed inpulverized heart tissues, which were freeze clamped afterassessment of in vivo cardiac function in fed mice (fordetails, see Supplementary Data).

Metabolic Flux Analysis in Ex Vivo Working HeartPerfused in Semi-Recirculating Working ModeMetabolic flux parameters were assessed in ex vivoworking perfused mouse hearts, as previously described indetail (18). Briefly, hearts isolated from fed animalswere perfused for 30 min under normoxia at fixed preload(15 mmHg) and afterload (50 mmHg) pressures withsemi-recirculating modified Krebs-Henseleit buffer con-taining a mix of substrates and hormones (11 mmol/Lglucose, 1.5 mmol/L lactate, 0.2 mmol/L pyruvate, and0.7 mmol/L palmitate bound to 3% dialyzed albumin,50 mmol/L carnitine, 0.8 nmol/L insulin, and 5 nmol/Lepinephrine). These perfusion conditions were used for allmouse groups in order to compare the intrinsic capacityof the heart to metabolize a fixed concentration of glucoseand palmitate, which we assessed by replacing, in anygiven perfusion, unlabeled palmitate or glucose with[U-13C6]glucose or [U-

13C16]palmitate, at 50% and 25% initialmolar percent enrichment, respectively. It is notewor-thy that while substrate concentrations are withinthe physiological level, they do not, however, mimicthose found in the diabetic state. Functional parameterswere continuously monitored throughout the perfusion(iox2 Data Acquisition System; emka TECHNOLOGIES).At the end of the perfusion, hearts were freeze clamped

with metal tongs chilled in liquid nitrogen and storedat280°C for further analysis. Using gas chromatography–mass spectrometry, we determined the 13C-labeling pat-tern of 1) lactate and pyruvate in influent and effluentperfusates and 2) tissue citrate. The following flux pa-rameters were calculated: 1) the efflux rate of labeledlactate and pyruvate arising from cytosolic glycolysisof exogenous [U-13C6]glucose and 2) the relative con-tribution of exogenous [U-13C6]glucose versus [U-

13C16]palmitate to mitochondrial acetyl CoA synthesis for cit-rate formation.

Statistical AnalysisData are reported as the mean 6 SE, and the statisticaldifference between groups was tested by two-way ANOVA,followed by a Bonferroni selected-comparison test to com-pare the respective source of variation (genotype and treat-ment) using GraphPad Prism software. When a significantinteraction was observed, the following groups were com-pared using one-way ANOVA followed by Bonferroni se-lected-comparison test (MK2+/+-vehicle vs. MK2+/+-STZ,MK22/2-vehicle vs. MK22/2-STZ, and MK22/2-STZ vs.MK2+/+-STZ). A P value ,0.05 was considered to be sta-tistically significant.

RESULTS

MK2 Deletion Prevents Diabetes-Induced CatabolicState and Improves Whole-Body IRWe first characterized the impact of our model of diabeteson systemic and metabolic parameters. Since MK2+/+ andMK22/2 vehicle-treated mice showed similar values for allanthropometric (Fig. 1) and glucose-related parameters(Fig. 2), these mice will be referred to as control mice,unless otherwise specified.

Figure 1—MK2 deletion prevents diabetes-induced catabolic state. A: Weekly calculated cumulative caloric intake: cumulative food intake(in g) from time 0 multiplied by 3.02 (in kcal/g) referring to metabolizable energy in the diet used. B: Weekly cumulative weight gainexpressed in percent from time 0. C and D: Glycolytic skeletal muscle (gastrocnemius) and epididymal WAT (eWAT) mass at week 15.Data are reported as the mean6 SE. n = 6–8. *P< 0.05, **P< 0.01, ***P< 0.001 vs. MK2+/+-vehicle; $P< 0.05, $$P< 0.01, $$$P< 0.001for MK22/2-STZ vs. MK2+/+-STZ. Two-way ANOVA with Bonferroni post-test.

diabetes.diabetesjournals.org Ruiz and Associates 383

Induction of diabetes by multiple low-dose injectionsof STZ in MK2+/+ mice (MK2+/+-STZ) led to a progressiveincrease in cumulative caloric intake, which reached sig-nificance at week 12 (Fig. 1A). This protocol also pro-voked a significant decrease in cumulative weight gain(14%; Fig. 1B), body weight (11%; Supplementary Table1), gastrocnemius (26%; Fig. 1C), and epididymal whiteadipose tissue (WAT; 3.6-fold; Fig. 1D) masses. In con-trast, MK22/2-STZ mice displayed values similar to thoseof control mice for each of these parameters.

We next evaluated metabolic parameters in 5-h fastedmice. Compared with control mice, MK2+/+-STZ miceshowed a 50% decrease in plasma insulin levels (Fig.2A), which was associated with an increase in blood glu-cose level (3.6-fold; Fig. 2B) and HbA1c level (2.5-fold; Fig.2C). MK22/2-STZ and MK2+/+-STZ mice showed similarplasma insulin levels at 15 weeks (Fig. 2A). In contrast,whereas glucose levels (Fig. 2B) were initially similar inMK22/2-STZ and MK2+/+-STZ mice, these values di-verged over time, and at 15 weeks the glucose levels inMK22/2-STZ mice were lower (23%) than those in theMK2+/+-STZ mice. However, these values remained two-fold greater than those for controls. Furthermore, HbA1c

levels in the MK22/2-STZ mice were also lower than inthe MK2+/+-STZ mice at 15 weeks (Fig. 2C).

To gain further insight into the differences betweenMK2+/+-STZ and MK22/2-STZ mice, we performed an OGTTafter a 16-h fast. Both MK2+/+-STZ and MK22/2-STZ mice

demonstrated a similar rapid and sustained increase inglucose level, resulting in a 2.5-fold greater area under thecurve (AUC) for glucose levels compared with control mice(Fig. 2D). This was consistent with the lower insulin levelsobserved in these animals (Fig. 2E). We next examined in-sulin sensitivity using an ipITT after a 5-h fast. As shown inFig. 2F, MK22/2-STZ mice displayed a significantly lowerAUC for glucose levels compared with MK2+/+-STZ mice(236%), suggesting an improvement in insulin sensitivity.

When these tests were assessed, we observed thatinitial glycemia was lower in STZ mice after 16 h offasting than after 5 h of fasting (Supplementary Fig. 2A).The reduction in glucose levels observed in MK22/2-STZmice compared with MK2+/+-STZ mice after 5 h of fasting(219.5%) was exacerbated after 16 h of fasting (236%)(Supplementary Fig. 2A). This reduction was paralleled bydecreased liver PEPCK expression (Supplementary Fig.2B), suggesting reduced hepatic glucose production.

MK2 Deletion Abrogates Diabetes-Induced CardiacDysfunction and Molecular ChangesNext, we assessed diabetes-induced cardiac alterationsat the functional and molecular levels. Hemodynamicanalyses using a Millar Mikro-Tip catheter revealed thatMK2+/+-STZ mice showed signs of cardiac dysfunctionin vivo, especially diastolic dysfunction (Fig. 3) with preservedsystolic function and hemodynamic parameters (Supple-mentary Table 1), as reflected by a significant increase in

Figure 2—MK2 deletion reduces IR (whole body) despite sustained hyperglycemia and hypoinsulinemia. A: End point (week 15) plasmainsulin level. B: Weekly fasting blood glucose levels (n = 6–8/group). C: Fasted glycated hemoglobin levels (n = 3–5). D: OGTT (glucosebolus 2 g/kg) results assessed at week 9 on fasted mice (16 h) and AUC analysis (n = 6–8). E: Insulin plasma level during the OGTT (4–5 bygroup and by time). F: ipITT (insulin 0.6 units/kg) assessed at week 10 on fasted mice (5 h) and AUC analysis (n = 6–7 by group and by time).Data are reported as the mean 6 SE. *P < 0.05, **P < 0.01, ***P < 0.001 vs. MK2+/+-vehicle; $P < 0.01, $$P < 0.01, $$$P < 0.001 forMK22/2-STZ vs. MK2+/+-STZ. Two-way ANOVA with Bonferroni post-test.

384 MK2 Deletion Improves Diabetic Cardiomyopathy Diabetes Volume 65, February 2016

left ventricular minimum diastolic pressure (Fig. 3A),mean diastolic pressure (Fig. 3B), and end-diastolic pres-sure, although the latter did not reach significance (P =0.1; Fig. 3C). While MK2+/+-STZ mice had a heart weight-to-body weight ratio similar to that of controls (Supple-mentary Table 1), these mice showed an eightfoldincrease in the ratio of b-myosin heavy chain (MHC) toa-MHC mRNA (Fig. 3D) (24). Furthermore, there was a sig-nificant decrease in SERCA2a (251%, Fig. 3E) as well as inphospholamban (PLB) phospho-Ser16 (247%, Fig. 3E) immu-noreactivity in MK2+/+-STZ mice, which suggest decreasedSERCA activity (25). There were, however, no changes in eithertranscript level or immunoreactivity for markers of endo-plasmic reticulum stress such as ATF6, GRP78, or CHOP(Supplementary Fig. 3) (26). Finally, consistent with thepreviously reported increase in p38MAPK signaling in di-abetic hearts (10), and in pathological cardiac remodeling(27), the phosphorylation of p38MAPK and MK2 wasincreased in MK2+/+-STZ mice by 3.5-fold (Fig. 4A) and3-fold (Fig. 4B), respectively, compared with controlmice. Moreover, diabetes did not alter total p38 andMK2 immunoreactivity (Fig. 4) or the basal expressionof proteins involved in insulin signaling, namely insulinreceptor substrate 1 (IRS-1), AKT, and the protein-tyrosinephosphatase 1B, a negative regulator of insulin signaling(Supplementary Fig. 4). Moreover, in the absence of a prior

insulin injection, IRS-1 phosphorylation (on serine 307)was undetectable, whereas AKT phosphorylation was sim-ilar among the four groups (Supplementary Fig. 4).

MK22/2-STZ mice were protected from all the afore-mentioned diabetes-induced cardiac alterations andshowed values similar to their control counterparts forall functional parameters (Fig. 3A–C and SupplementaryTable 1). In addition, the b-MHC–to–a-MHC ratio (Fig.3D), SERCA2a expression, and PLB phosphorylation(Fig. 3E) in MK22/2-STZ mice were similar to those oftheir control counterparts. As expected, total and phos-phorylated MK2 immunoreactivities were undetectable incontrol and diabetic MK22/2 mice. As MK2 stabilizesp38a (28), total and phosphorylated p38 immunoreactiv-ity was reduced (287% and 272%, respectively) inMK22/2 mice (Fig. 4).

MK2 Deletion Protects Against Diabetes-InducedSystemic and Cardiac Lipid PerturbationsGiven our findings that the benefits of MK2 abrogationon diabetes-induced cardiac alterations 1) occurred de-spite sustained hyperglycemia and 2) were associatedwith preserved WAT mass, we hypothesized that thecardioprotective effect of knocking out MK2 could beassociated with the normalization of diabetes-inducedsystemic and/or cardiac lipid alterations. As expected, at15 weeks, MK2+/+-STZ mice showed elevated circulating

Figure 3—MK2 deletion prevents diabetes-induced cardiac dysfunction. Diastolic functional parameters determined using a Millar catheterin fed 20-week-old mice (n = 4–8) highlighted by minimal pressure (A), mean diastolic pressure (B), and end diastolic pressure (C ).D: MHC isoform mRNA expression (n = 4–8/group) assessed in whole extract of nonperfused hearts from fed mice and normalizedto cyclophilin A (CycA) expression. E: Representative images (left panel) and quantitation (histograms) of SERCA2a, phospholamban(P-PLB [P-Ser16-PLB]), and PLB immunoreactivity. Immunoreactivity was assessed in whole extracts of nonperfused hearts from fedmice (n = 4/group) and normalized to b-actin immunoreactivity. AU, arbitrary unit; Veh, vehicle. Data are reported as themean6 SE. MK2+/+-STZ vs.MK2+/+-vehicle, *P < 0.05, **P < 0.01; MK22/2-STZ vs. MK2+/+-STZ, $P < 0.05, $$P < 0.01. Two-way ANOVA with Bonferroni post-test.

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plasma FFA levels (44%; Fig. 5A) and b-hydroxybutyrate(b-HB) levels (384%; Fig. 5B) compared with their controlcounterparts. Remarkably, FFA (Fig. 5A) and b-HB (Fig.5B) levels in MK22/2-STZ mice were similar to those incontrols. We then examined plasma AC levels, which aremonitored routinely when screening for defects in tissueFA b-oxidation (29) and have been found to be associatedwith IR and diabetes (30). The plasma levels of short-chain ACs and medium-chain ACs were similar amongall four mouse groups (Supplementary Fig. 5). However,MK2+/+-STZ mice displayed a significant increase in long-chain ACs (LCACs) (Fig. 5C), namely C14:2-carnitines(63%), C18-carnitines (238%), C18:1-carnitines (61%),and C18:2-carnitines (200%). In contrast, MK22/2-STZmice maintained plasma LCAC levels that were similarto those in their control counterparts.

We next ascertained whether these systemic changes inlipid metabolism would be paralleled by changes in theircardiac metabolism by conducting metabolic flux studiesin ex vivo working hearts using 13C-labeled glucose andpalmitate. It is noteworthy that functional parametersrecorded during the perfusions demonstrated that alter-ations observed in vivo (Fig. 3 and Supplementary Table1) were also present in ex vivo working heart prepara-tions. Indeed, compared with control mice, MK2+/+-STZmice displayed diastolic dysfunction (220% for minimumchange in pressure over time (dP/dt; Fig. 6B), systolicdysfunction (218% and 222%, respectively, for maximalpressure and maximum dP/dt; Fig. 6C and D), and hemo-dynamic alterations (222% for stroke volume; Fig. 6E).

MK22/2-STZ mice were, however, protected against allthese diabetes-induced cardiac functional alterations.

Figure 7 and Supplementary Fig. 6A and B report met-abolic flux parameters from these hearts. First, flux re-lated to glucose metabolism did not differ significantlyamong groups. These fluxes include rates of lactateand pyruvate efflux formed from glycolysis of exogenous[U-13C6]glucose (Supplementary Fig. 6A) as well as the relativecontribution of glucose to mitochondrial acetyl-CoA for-mation through pyruvate decarboxylation (Supplemen-tary Fig. 6B). Moreover, because myocardial oxygenconsumption (MVO2) was not significantly differentamong the four groups (in mmol/min, MK2+/+-vehicle1.50 6 0.20, MK2+/+-STZ 1.02 6 0.23, MK22/2-vehicle1.11 6 0.11, and MK22/2-STZ 1.46 6 0.30), this wouldalso suggest a similar absolute flux of glucose to acetyl-CoA(calculated from the stoichiometric relationship betweenMVO2 and citrate formation from glucose as determinedfrom the measured flux ratio [31]). We did not assess themetabolism of other carbohydrates, such as exogenous lac-tate or pyruvate. The contribution of exogenous palmitateto acetyl-CoA via b-oxidation was, however, significantlyincreased (46%) in MK2+/+-STZ mice compared with theircontrol counterparts (Fig. 7A). In contrast, in MK22/2-STZmice b-oxidation rates were similar to MK22/2-vehicle orMK2+/+-vehicle mice.

The cardiac transcript levels for genes involved incarbohydrates and FA metabolism, which we assume toreflect protein levels, were consistent with the observedchanges in cardiac metabolic flux parameters. Specifically,

Figure 4—Diabetes activates the p38 pathway in the heart. A: Representative images (top panel) and quantitation (histograms) ofphospho-p38 (P-P38 [P-Thr180/Tyr182-p38]) and total p38 immunoreactivity. B: Representative images (top panel) and quantitation(histograms) of phospho-MK2 (P-MK2 [P-Thr222-MK2]) and total MK2 immunoreactivity. Both p38 and MK2 immunoreactivity wereassessed in whole extracts of nonperfused hearts from fed mice. Data are reported as the mean 6 SE (n = 4) and normalized to b-actinimmunoreactivity. AU, arbitrary unit; ND, not determined; Veh, vehicle. MK22/2-STZ vs. MK2+/+-STZ, *P < 0.05, $P < 0.05, $$$P < 0.001;MK22/2-vehicle vs. MK2+/+-vehicle, &&&P < 0.001. Two-way ANOVA with Bonferroni post-test.

386 MK2 Deletion Improves Diabetic Cardiomyopathy Diabetes Volume 65, February 2016

in MK2+/+-STZ mice, although there was a significant de-crease in the abundance of mRNA for GLUT1 (226%), theother measured markers of glucose transport and glycol-ysis (GLUT4, PFK1, and PFK2) did not differ significantlyfrom those of MK2+/+-vehicle mice (Supplementary Fig.6C and D). Moreover, MK2+/+-STZ mice showed an in-crease in transcript levels for the FA transporters (CD3625%, CPT1b 27%) and oxidation (long-chain acyl-CoA de-hydrogenase 25%; Fig. 7B), as well as for the negativeregulator of mitochondrial pyruvate dehydrogenasePDK4 (fourfold; Supplementary Fig. 6D). In contrast, foreach of these markers, MK22/2-STZ and MK22/2-vehiclemice did not differ significantly.

We also investigated the mechanisms involved inmitochondrial FA oxidation, namely phosphorylation ofAMPK and its downstream target acetyl-CoA carboxylase(ACC), transcript levels for PPARa, peroxisome proliferator–activated receptor a coactivator (PGC-1a), and uncouplingprotein 3 (UCP3), as well as the level of mitochondrialelectron transport chain (ETC) proteins. Althoughhearts from MK2+/+-STZ mice displayed significantlyincreased levels of AMPK phosphorylation (330%; Fig.7C) and UCP3 mRNA (210%, Fig. 7E), the abundanceand phosphorylation of ACC (Fig. 7D), PPARa, andPGC-1a mRNA (Supplementary Fig. 7B), as well as immu-noreactivity for components of the mitochondrial ETC(Supplementary Fig. 7A) remained unchanged. In con-trast, MK22/2-STZ mice did not display any significantchanges compared with their control counterpart inAMPK phosphorylation and transcript levels of UCP3.Moreover, as observed in MK2+/+-STZ mice, ACCphosphorylation (Fig. 7D), PPARa or PGC-1a mRNAexpression, and mitochondrial ETC protein levels

(Supplementary Fig. 7A and B) were unchanged inMK22/2-STZ mice.

Finally, MK2+/+-STZ mice demonstrated a 38% in-crease in cardiac TG content in comparison with theirvehicle controls and MK22/2-STZ mice (Fig. 8A). To fur-ther explore potential mechanisms underlying this TGaccumulation, we assessed the expression of enzymes in-volved in TG synthesis (GPAT1) or hydrolysis (ATGL andHSL) by immunoblotting, as well as transcript levels ofPPARd/b, which has been inversely associated with TGcontent in diabetic hearts (32). Although GPAT1, ATGL,and HSL immunoreactivity were unchanged, we foundsignificant increases in the phosphorylation of HSL onserine 565 (405%; Fig. 8B), a specific target of AMPKthat inhibits HSL activity (33), as well as a reduction ofPPARd/b mRNA level (258%; Supplementary Fig. 7B) inMK2+/+-STZ mouse hearts, but not in MK22/2-STZ mousehearts.

Collectively, all measured parameters relevant to FAmetabolism convey a similar message, as follows: heartsfrom MK2+/+-STZ mice show enhanced exogenous FAoxidation and esterification for storage, whereas theirMK22/2-STZ counterparts showed no sign of altered FAmetabolism, which is consistent with the lack of changesin systemic FAs in these mice.

DISCUSSION

Our results demonstrate that whole-body MK2 deletionin mice protects against diabetes-induced systemic andcardiac metabolic perturbations, as well as against cardiacdysfunction, despite sustained hyperglycemia. Specifically,the improvement in cardiac function was associated withnormalization of diabetes-induced 1) cardiac molecular

Figure 5—MK2 deletion abrogates diabetes-induced systemic lipid perturbations. Fast (5-h) end-point (week 15) plasma FFA levels (n =6–8 by group) (A), ketonemia levels (indicated by b-HB, n = 5–6) (B), and plasma LCACs (C14, C14:2, C16, C16:2, C18, C18:1, C18:2,C18:3) (C ) reflecting the b-oxidation function (n = 4 by group). Data are reported as the mean 6 SEM. MK2+/+-vehicle vs. MK2+/+-STZ,*P < 0.05, **P < 0.01; MK22/2-STZ vs. MK2+/+-STZ, $P < 0.05, $$P < 0.01. Two-way ANOVA with Bonferroni post-test.

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changes, including proteins involved in maintaining cal-cium homeostasis; 2) systemic lipid abnormalities; and3) cardiac lipid abnormalities.

In this study, diabetes was induced by repeatedadministration of low doses of STZ to mice fed a standardchow diet. MK2+/+-STZ mice showed a 50% decrease inplasma insulin levels in addition to IR, hyperglycemia,increased HbA1c levels, and augmented ketonemia. Thesemice also displayed a loss of WAT and skeletal musclemass, as well as loss of body weight despite an increasein caloric intake, which is indicative of a catabolic state.These phenotypic characteristics differ to some extent,particularly insulin secretion, from those induced byhigh doses of STZ, leading to almost total suppressionof insulin secretion and type 1 diabetes (34), or by re-peated low doses of STZ combined with a high-fat diet,which lead to IR and hyperinsulinemia, mimicking type 2diabetes (35). Hence, our model seems to share charac-teristics of type 1 diabetes (catabolic state and increasedketonemia) and advanced type 2 diabetes (IR and 50%insulin level reduction).

Given that the main objective of this study was to testthe impact of a whole-body MK2 deletion on diabetes-induced cardiac dysfunction, it was crucial to ascertainthat our diabetes model develops cardiac abnormalities aspreviously described (36). Consistent with other model ofdiabetes, MK2+/+-STZ mice displayed in vivo diastolic dys-function, and changes in the expression of b- and a-MHCisoforms. Cardiac dysfunction was exacerbated ex vivo inisolated working heart preparations, where increased di-astolic as well as systolic dysfunctions were observed.The latter finding suggests the presence of systemic ho-meostatic compensatory mechanisms (e.g., neuronal or

hormonal influences), which are present in the intactanimal but are absent in ex vivo preparations. Consistentwith the observed diastolic dysfunction, MK2+/+ diabeticmice displayed significant changes in proteins regulatingcardiac excitation-contracting coupling, namely lowerSERCA2a immunoreactivity and activity, as reflected byreduced PLB phosphorylation, suggesting disturbancesin Ca2+ reuptake during diastole (37). Remarkably,MK22/2 mice were protected against diabetes-inducedcardiac dysfunction concomitant with normalization ofSERCA2a expression and PLB phosphorylation. The lat-ter finding is consistent with the recently proposed roleof the p38-MK2/3 axis in regulating cardiomyocyte func-tion through SERCA2a modulation (15). In this regard,results from our study demonstrate a specific role ofMK2, which may act directly or through its capacity tomodulate the abundance of p38a (28,38). Interestingly,the specific inhibition of p38aMAPK, but not p38bMAPK,was recently shown (39) to enhance diastolic Ca2+

uptake through increased PLB phosphorylation on theresidue Ser16.

Taken together, the results from our study, in additionto those of others (15–17), demonstrate that suppressingMK2 activity has beneficial consequences for the heartunder several pathological conditions. This study specifi-cally substantiates its benefits against diabetes-inducedmetabolic alterations and cardiac dysfunction. However,similar to other studies, our mouse model is a whole-bodyMK2 knockout; thus, we were unable to discriminate be-tween its cardiac and systemic effects. We observed addi-tional effects of MK2 deletion in diabetic mice, beyondthose relevant to cardiac calcium handling, which maydirectly or indirectly preserve cardiac function. Indeed,

Figure 6—MK2 deletion prevents diabetes-induced dysfunction in ex vivo working hearts. Functional parameters of ex vivo hearts from fed20-week-old mice (n = 7–12). A and B: Diastolic function represented by minimal pressure and minimal change in pressure over time(minimum dP/dt [dP/dt min]). C and D: Systolic function represented by maximal pressure and maximal change in pressure over time(maximum dP/dt [dP/dt max]). E and F: Hemodynamic parameters, namely stroke volume and cardiac output. Data were acquired over aperiod of 20–25 min. Data are reported as the mean 6 SE. MK22/2-STZ vs. MK2+/+-vehicle, *P < 0.05, **P < 0.01; MK22/2-STZ vs.MK2+/+-STZ, $P < 0.05. Two-way ANOVA with Bonferroni post-test.

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we showed that major diabetes-induced systemic and car-diac lipid abnormalities, which have been previously associ-ated with cardiac dysfunction in both patients with diabetes(40) and several experimental diabetes models (41,42),were abrogated in MK22/2-STZ mice. At the systemiclevel, MK2+/+-STZ mice displayed increased levels ofplasma FFAs in combination with reduced WAT mass,suggesting an augmented WAT lipolysis (43). Further-more, our finding of a selected increase in plasma LCACs,but not medium-chain ACs or short-chain ACs, suggeststhat FA cellular overload, namely increased FA cellularuptake and activation to acyl-CoA, exceeds the capacityof mitochondria to take up acyl-CoA for subsequent FAoxidation. The latter could be the consequence of a mis-match between expression or activity of the mitochondri-al FA transporters CPT1, which converts LC-acyl-CoA toLCACs, and CPT2, which converts LCACs back to LC-acyl-CoA. While the increased levels of plasma LCACs reflectdisturbances from multiple organs, our results in the heartsupport the proposed scenario. Indeed, in the MK2+/+-STZmice, cardiac transcript levels for the cellular long-chainFA (LCFA) transporter CD36 as well as CPT1 were in-creased, whereas those of CPT2 were unchanged. Alongwith this mismatch between LCFA uptake into cells and

mitochondria, other data indicate that diabetic MK2+/+-STZ mouse hearts are coping with the excessive LCFAuptake by shuttling proportionally more FAs to mito-chondrial oxidation, while restricting TG hydrolysis. Anaugmented relative contribution of mitochondrial FA oxi-dation to acetyl-CoA generation for citrate formation issupported by metabolic flux measurements in ex vivoworking hearts and by expression data for proteins in-volved in FA oxidation or its regulation. Moreover, whileTG accumulation occurs without changes in the abundanceof enzymes involved in TG synthesis (DGAT1) or hydroly-sis (HSL and ATGL), there was, however, increased AMPK-dependent phosphorylation of HSL, which is known toinhibit its activity and is consistent with increased TGaccumulation (33). These changes were also consistentwith a decrease in PPARb/d expression, which was previ-ously shown to parallel cardiac TG accumulation (44). Thefact that the MVO2 tended to be slightly decreased indiabetic MK2+/+ hearts suggests, however, that globallymitochondrial oxidative metabolism, which encompassesabsolute b-oxidation flux rate from FAs, both exogenouslysupplied and endogenously formed from TG, wouldalso be decreased. All these metabolic changes, togetherwith the observed cardiac dysfunction, suggest that the

Figure 7—MK2 deletion attenuates the diabetes-induced increase in palmitate oxidation in ex vivo working hearts and prevents theincrease in expression of markers of FA metabolism. A: Relative contribution of exogenous palmitate to acetyl-CoA formation for citratesynthesis (n = 4–6). B: mRNA levels of FA transport markers (CD36, CPT1b, and CPTII) and FA oxidation (medium-chain acyl-CoAdehydrogenase [MCAD] and long-chain acyl-CoA dehydrogenase [LCAD]) (n = 4–8) assessed in whole extract of nonperfused heartsfrom fed mice and normalized to cyclophilin A (CycA) expression. C: Representative images (left panel) and quantitation (histograms)of phospho-AMPK (P-AMK [P-Thr172-AMPK]) and AMPK immunoreactivity. D: Representative images (left panel) and quantitation(histograms) of phospho-ACC (P-ACC [P-Ser79-ACC]) and ACC immunoreactivity. Immunoreactivity was assessed in whole extract ofnonperfused hearts from fed mice (n = 4/group) and normalized to b-actin immunoreactivity. AU, arbitrary unit; Veh, vehicle. E: mRNAlevels of the uncoupling protein UCP3 (n = 4–8) assessed in whole extract of nonperfused hearts from fed mice and normalized toCycA expression. Data are mean 6 SE. MK22/2-STZ vs. MK2+/+-vehicle, *P < 0.05, **P < 0.01. Two-way ANOVA with Bonferronipost-test.

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metabolic flexibility of diabetic MK2+/+ hearts is impaired,while being preserved in MK22/2 diabetic hearts. Thisconclusion needs, however, to be further substantiatedby testing the metabolic and functional response ofthese diabetic hearts after a challenge such as high work-load. Further studies are also required to determine thespecific roles and mechanisms by which MK2 modulatesdiabetes-induced systemic versus cardiac lipid metabo-lism. Although PPARa, PGC-1a, and/or mitochondrialbiogenesis are known to be factors determinant for lipidmetabolism and have been reported to be part of diabetes-induced cardiac metabolic changes (45), our mRNA ex-pression data do not support their involvement in ourdiabetes model, or as a MK2 target. Although additionaldata on protein expression and activity would be neededto ascertain this conclusion, there are several others fac-tors that ought to be considered as potential, direct orindirect, players such as IR. Whole-body insulin sensi-tivity has been linked to perturbations in systemic lipidmetabolism as well as ectopic lipid deposition, includingthe heart (46). Although we did not detect any sign ofcardiac IR under the basal state, as reflected by undetect-able IRS-1 phosphorylation levels (on serine 307) and un-changed AKT phosphorylation, additional studies shouldinvestigate the role of MK2 in insulin action/signaling indiabetes.

IR alters fat deposition in WAT and liver (47). Theimproved insulin sensitivity in MK22/2-STZ mice mayexplain, at least in part, the observed increase of WATmass and subsequently the decrease of plasma FFA lev-els compared with MK2+/+-STZ mice, as well as the

small, but significant, reduction in fasting hyperglycemia,which may result from normalization of the diabetes-induced increase in liver PEPCK expression in MK22/2-STZmice (this study) and gluconeogenesis by MK2 inhibi-tion (14). It is noteworthy that our results differ tosome extent from those of de Boer et al. (48), whoshowed that MK2 deficiency reduces insulin sensitivityin high-fat diet–fed mice. Although in the latter studymodel mice are hyperinsulinemic, in contrast to ourMK22/2-STZ mice, which are hypoinsulinemic, insulinlevel is unlikely to be the sole explanation. Indeed,expressing dominant-negative MK2 or administrationof an allosteric MK2 inhibitor in ob/ob mice, a hyper-insulinemic mouse model, also improved liver andwhole-body insulin sensitivity, respectively (14). Thereby,other factors are likely to be involved. In this regard,another mechanism that may underlie the beneficialeffect of MK2 deletion on insulin sensitivity as well assystemic and cardiac lipid metabolism is an attenuatedinflammatory response (47), although this remains tobe further investigated. Activation of the p38MAPK/MK2 axis has been linked to a proinflammatory re-sponse (49), and MK22/2 mice were shown to be moreresistant to stress and endotoxic shock (19). A recent studyprovides valuable insight into the differential effect ofp38MAPK versus MK2 inhibition in modulating proinflam-matory signaling pathways. In both cases, there is inhibitionof p38/MAPK signaling (50). However, inhibition of p38,without discrimination of the isoform, induces a sustainedactivation of c-Jun N-terminal kinase and nuclear factor-kB,whereas the inhibition of MK2 does not (50).

Figure 8—MK2 deletion abrogates diabetes-induced cardiac TG accumulation. A: TG content in nonperfused cardiac tissue from fed micewas estimated by global LCFA accumulation in TGs (n = 5–8). B: Representative images (top panel) and quantitation (histograms) ofimmunoreactivity of GPAT1, ATGL, HSL, and phospho-HSL (P-HSL [P-Ser565-HSL]) immunoreactivity. Immunoreactivity was assessed inwhole extracts of nonperfused hearts from fed mice (n = 4/group) and normalized to b-actin immunoreactivity. AU, arbitrary unit; Veh,vehicle. Data are reported as the mean6 SE. MK22/2-STZ vs. MK2+/+-vehicle, *P < 0.05; MK22/2-STZ vs. MK2+/+-STZ, $$P < 0.01. Two-way ANOVA with Bonferroni post-test.

390 MK2 Deletion Improves Diabetic Cardiomyopathy Diabetes Volume 65, February 2016

In summary, the current study demonstrates that thedeletion of MK2 prevents diabetes-induced cardiac dys-function, an effect that may involve alterations in theexpression and activation of SERCA2a and PLB, proteinsinvolved in maintaining cardiac calcium homeostasis. Ourresults also identify other effects of MK2 deletion ondiabetes-induced lipid perturbations both at the systemicand cardiac level, which could also protect cardiac func-tion. Future studies using mice with a cardiomyocyte-specific MK2 deletion are, however, needed to dissect 1)the molecular mechanisms by which MK2 modulate car-diac lipid metabolism and cardiac function and 2) theprimary compartments in which these effects of MK2occur, namely heart versus other tissues. Takenaltogether, our findings suggest that MK2 inhibitionmay represent a new strategy to protect the myocardiumfrom diabetes-induced alterations in systemic and cardiacFA metabolism, as well as cardiac dysfunction.

Acknowledgments. The authors thank Anik Forest, Research Centre,Montreal Heart Institute, for helpful comments for liquid chromatography–massspectrometry methodology; Marie Eve Rivard, Isabelle Robillard-Frayne, andNayla El Zyr, Research Centre, Montreal Heart Institute, the animal facility staff,for technical assistance; and France Thériault, Research Centre, Montreal HeartInstitute, for editorial and secretarial assistance.Funding. This work was supported by the Canadian Institutes of Health Re-search (grant 9575 to C.D.R.) and the Heart and Stroke Foundation of Canada(grant G-14-0006060 to B.G.A.).

The funders played no role in the study.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. M.R. contributed to the design of the project,performed the experiments, analyzed all of the data, and wrote, reviewed, andedited the manuscript. L.C. and B.G.A. contributed to the design of the projectand wrote, reviewed, and edited the manuscript. D.L., V.H., C.M., J.T.L., M.-A.G.,B.B., and C.D. performed the experiments. A.C.C. contributed to the design of theproject. M.G. provided material essential for the study. C.D.R. contributed to thedesign of the project and wrote, reviewed, and edited the manuscript. C.D.R. isthe guarantor of this work and, as such, had full access to all the data in thestudy and takes responsibility for the integrity of the data and the accuracy of thedata analysis.

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