IntroductionMetabolic reprogramming from glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation may mediate macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. We hypothesized that changes in cardiac macrophage glucose metabolism would reflect polarization status after myocardial infarction (MI), ranging from the early inflammatory phase to the later wound healing phase. MethodsMI was induced by permanent ligation of the left coronary artery in adult male C57BL/6J mice for 1 (D1), 3 (D3), or 7 (D7) days. Infarct macrophages were subjected to metabolic flux analysis or gene expression analysis. Monocyte versus resident cardiac macrophage metabolism was assessed using mice lacking the Ccr2 gene (CCR2 KO).ResultsBy flow cytometry and RT-PCR, D1 macrophages exhibited an M1 phenotype while D7 macrophages exhibited an M2 phenotype. Macrophage glycolysis (extracellular acidification rate) was increased at D1 and D3, returning to basal levels at D7. Glucose oxidation (oxygen consumption rate) was decreased at D3, returning to basal levels at D7. At D1, glycolytic genes were elevated (Gapdh, Ldha, Pkm2), while TCA cycle genes were elevated at D3 (Idh1 and Idh2) and D7 (Pdha1, Idh1/2, Sdha/b). Surprisingly, Slc2a1 and Hk1/2 were increased at D7, as well as pentose phosphate pathway (PPP) genes (G6pdx, G6pd2, Pgd, Rpia, Taldo1), indicating increased PPP activity. Macrophages from CCR2 KO mice showed decreased glycolysis and increased glucose oxidation at D3, and decreases in Ldha and Pkm2 expression. Administration of dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, robustly decreased pyruvate dehydrogenase phosphorylation in the non-infarcted remote zone, but did not affect macrophage phenotype or metabolism in the infarct zone.DiscussionOur results indicate that changes in glucose metabolism and the PPP underlie macrophage polarization following MI, and that metabolic reprogramming is a key feature of monocyte-derived but not resident macrophages.

Important adverse changes in cardiac energy metabolism occur after ischemia/reperfusion injury (I/R), contributing to the worsening of cardiac function and development of heart failure. We recently showed that leptin, via its actions on the central nervous system (CNS), improves left ventricular (LV) function in a model of heart failure induced by permanent ligation of the left anterior descending coronary artery (LAD). In the present study, we examined whether the CNS‐ effects of leptin protect against myocardial I/R injury, and if chronic intracerebroventricular (ICV) leptin infusion improves cardiac substrate utilization, assessed by markers of myocardial fatty acid (FA) and glucose oxidation, NAD+/NADH redox state and plasma levels of β‐hydroxybutyrate (β‐HOB). Male Wistar rats were instrumented with an ICV cannula in the brain lateral ventricle. After recovery and baseline assessment of cardiac function by echocardiography (ECHO), myocardial I/R was induced by temporary (60 min) ligation of the LAD. Vehicle (saline, 0.5 µL/hr) or leptin (0.62 µg/hr) was infused chronically for 28 days starting 20 min after reperfusion using osmotic minipumps connected to the ICV cannula. ECHO assessment of cardiac function was performed weekly. At the end of week 4, +dP/dtmax was accessed by LV catheterization. Hearts and plasma samples were collected for evaluation of CD36, PPAR‐δ, PDK4 and p‐PDH1/PDH1 by western blot and cardiac NAD+/NADH ratio and plasma levels of β‐OHB were measured by ELISA. Our results showed that ICV leptin treatment improved cardiac function as evidenced by increased ejection fraction 4 weeks after I/R (46±3 vs. 26±3 %) and +dP/dtmax (10387±1686 vs. 5022±442 mmHg/s) when compared with vehicle‐treated rats. ICV leptin infusion also significantly increased cardiac protein expression of CD36 (1.4±0.1 vs. 1.0±0.1 au), PPAR‐δ (1.8±0.1vs. 1.0±0.01 au), PDK4 (1.8±0.1 vs. 1.0±0.1 au) and p‐PDH1/PDH1 (2.2±0.3 vs. 1.0±0.2 au) when compared with vehicle‐treated animals. In addition, ICV leptin infusion increased cardiac NAD+/NADH ratio (6.8±2 vs. 1.02±0.3 pmol/µL) and reduced plasma levels of β‐OHB (18.2±3 vs. 62.8±7.0 nmol/µL). These results demonstrate that chronic ICV leptin infusion improves cardiac function following I/R injury and suggest that leptin’s CNS‐mediated cardioprotective effects may involve improved myocardial FA oxidation and NAD+/NADH redox state, reducing the reliance on ketone bodies (β‐OHB) as an energy source.

We previously demonstrated that central nervous system (CNS) administration of leptin improves left ventricular (LV) function after myocardial infarction through mechanisms that are poorly understood. Increased cardiac sympathetic activity is an important compensatory mechanism to maintain cardiac output after ischemic injury, and previous studies showed that centrally-administered leptin increases sympathetic outflow to various tissues. In the present study, we tested the hypothesis that leptin’s CNS-mediated cardioprotective effects are mediated via cardiac sympathetic nerves. Male and female Wistar rats (~8 weeks of age) were submitted to cervical ganglia denervation (CGx) and instrumented with an intracerebroventricular (ICV) cannula in the brain lateral ventricle. After recovery and baseline assessment of cardiac function by echocardiography (ECHO) and an exercise performance test (Vmax), myocardial ischemia/reperfusion (I/R) was induced by temporary (60 min) ligation of the left anterior descending coronary artery. Vehicle (saline, 0.5 μL/hr) or leptin (0.62 μg/hr) was infused chronically for 28 days starting 20 min after reperfusion using osmotic pumps implanted subcutaneously in the scapular region and connected to the ICV cannula. Cardiac function was assessed weekly by ECHO. At the end of week 4, a final Vmax was performed and +dP/dt max was accessed by LV catheterization using a Millar catheter. Despite ~70% reduction in cardiac tyrosine hydroxylase positive fibers compared to sham, CGx did not prevent leptin’s CNS-mediated cardioprotective effects as indicated by improved ejection fraction (55±1 vs. 29±3 %), global longitudinal strain (-19±2 vs. -3±2 %), +dP/dt max (10214±614 vs. 7174±716 mmHg/s) and exercise performance (1.3±14 vs. -46.3±14 Δ% compared to baseline before I/R) when compared with CGx vehicle-treated animals. CGx also did not prevent the effect of ICV leptin to reduce septal collagen deposition and heart weight/tibia length post-I/R. These results suggests that leptin, via its CNS actions, improves cardiac function and remodeling after I/R injury independent of cardiac sympathetic innervation.

Background Therapeutic strategies for preventing paradoxical reperfusion injury after myocardial ischemia are limited. We tested whether central nervous system actions of leptin induce important protective effects on cardiac function and metabolism after myocardial ischemia/reperfusion (I/R) injury, the role of cardiac sympathetic innervation in mediating these effects, and whether there are major sex differences in the cardioprotective effects of chronic central nervous system leptin infusion. Methods and Results Myocardial I/R was induced by temporary ligation of the left descending coronary artery in male and female Wistar rats instrumented with intracerebroventricular cannula in the lateral ventricle. Vehicle or leptin (0.62 μg/h) infusion was started immediately after reperfusion and continued for 28 days using osmotic minipumps connected to the intracerebroventricular cannula. Cardiac function was assessed by echocardiography, ventricular pressures, and exercise performance. Intracerebroventricular leptin treatment markedly attenuated cardiac dysfunction post‐I/R as evidenced by improved ejection fraction (56.7±1.9 versus 22.6%±1.1%), maximal rate of left ventricle rise (11 680±2122 versus 5022±441 mm Hg) and exercise performance (−4.2±7.9 versus −68.2±3.8 Δ%) compared with vehicle‐treated rats. Intracerebroventricular leptin infusion reduced infarct size in females, but not males, when compared with ad‐lib fed or pair‐fed saline‐treated rats. Intracerebroventricular leptin treatment also increased cardiac NAD + /NADH content (≈10‐fold) and improved mitochondrial function when compared with vehicle treatment. Cervical ganglia denervation did not attenuate the cardiac protective effects of leptin after I/R injury. Conclusions These data indicate that leptin, via its central nervous system actions, markedly improves overall heart function and mitochondrial metabolism after I/R injury regardless of sex, effects that are largely independent of cardiac sympathetic innervation.

We previously demonstrated that activation of the central nervous system (CNS) melanocortin system modulates sympathetic nerve activity (SNA) and blood pressure (BP) in obese Zucker rats and spontaneously hypertensive rats (SHRs). In the present study, we examined whether endogenous activation of the brain melanocortin-4 receptors (MC4R) contributes to the regulation of BP and metabolic function in adult male (24-26 weeks-old) obese and lean offspring from parents fed a normal (N) or high fat diet (H). The rats were implanted with telemetry probes to measure BP and heart rate (HR) 24-hrs/day, and an intracerebroventricular (ICV) cannula was placed into the lateral ventricle. After 1 week to recover from surgery and 2 days of control measurements, the MC4R antagonist (SHU-9119) was infused (1 nmol/h, ICV) for 7 days followed by a 7-day recovery period. MC4R antagonism markedly increased food intake in all groups; however, the increase was more pronounced in obese offspring from obese H-fed parents (HH), especially when analyzing the net cumulative increase in food intake during the 7 days of treatment (191±11 vs. 136±14, 65±18 and 72±12 g) compared with obese offspring from lean N-fed parents (NH), lean offspring from obese H-fed parents (HN) and lean offspring from lean parents (NN), respectively. This increased food intake was associated with more weight gain in HH compared with NH, HN and NN offspring (Δ: 92±12 vs. 81±17, 52±11 and 38±9 g). Plasma leptin and insulin levels were markedly increased in HH offspring compared to NH, HN and NN offspring. Compared to NH and NN offspring from lean parents, HH and HN offspring from obese parents exhibited higher baseline mean arterial pressure (125±2 and 122±3 vs. 118±1 and 113±3 mmHg) and greater reduction in BP during SHU-9119 infusion (average last 3 days of infusion: -14.3±0.1 and -5.6±0.5 vs. -9.8±0.2 and -3.4±0.2 mmHg, respectively). The reduction in HR during SHU-9119 infusion, however, was more pronounced in NH and NN groups compared with HH and HN offspring (average last 3 days of infusion: -55±2 and -43±1 vs. -34±2 and -34±1 bpm). These results suggest that endogenous activation of brain MC4R may contribute to the elevated BP in obese offspring but not lean offspring from obese parents.

We examined the impact of parental obesity on offspring blood pressure (BP) regulation and cardiovascular responses to stress. Offspring from normal (N) diet-fed C57BL/6J parents were fed either N (NN) or a high-fat (H) diet (NH) from weaning until adulthood. Offspring from obese H diet-fed parents were also fed N (HN) or H diet (HH). Body weight, calorie intake, and fat mass were measured at 22 wk of age when cardiovascular phenotyping was performed. Male and female HH offspring were 15% heavier than NH and 70% heavier than NN offspring. Male HH and HN offspring had elevated BP (121 ± 2 and 115 ± 1 mmHg, by telemetry) compared with male NH and NN offspring (108 ± 6 and 107 ± 3 mmHg, respectively) and augmented BP responses to angiotensin II, losartan, and hexamethonium. Male HH and HN offspring also showed increased BP responses to air-jet stress (37 ± 2 and 38 ± 2 mmHg) compared with only 24 ± 3 and 25 ± 3 mmHg in NH and NN offspring. Baseline heart rate (HR) and HR responses to air-jet stress were similar among groups. In females, BP and cardiovascular responses to stress were similar among all offspring. Male H diet-fed offspring from obese H diet-fed purinoreceptor 7-deficient (HH-P2X7R-KO) parents had normal BP that was similar to control NN-P2X7R-KO offspring from lean parents. These results indicate that parental obesity leads to increased BP and augmented BP responses to stress in their offspring in a sex-dependent manner, and the impact of parental obesity on male offspring BP regulation is markedly attenuated in P2X7R-KO mice.

Parental obesity contributes to diastolic dysfunction in young offspring (1–3 days after weaning) in a sex-dependent manner, as well as reduced cardiac SIRT3 expression and altered mitochondrial bioenergetics, resting Ca2+ levels, and reduced phospholamban protein levels.

Mitochondrial (MT) dysfunction plays an important role in the pathophysiology of myocardial ischemia/reperfusion injury (I/R). We recently showed that leptin, via its actions in the central nervous system (CNS), improves left ventricular (LV) function in a model of heart failure induced by permanent ligation of the left anterior descending coronary artery (LAD). In the present study, we examined if the CNS effects of leptin protect against myocardial ischemia/reperfusion (I/R) injury, and whether chronic intracerebroventricular (ICV) leptin infusion increases MT function and biogenesis in the non-infarcted area of the LV that is at risk but still viable. Male Wistar rats were instrumented with an ICV cannula in the brain lateral ventricle. After recovery and baseline assessment of cardiac function by echocardiography (ECHO), myocardial I/R was induced by temporary (60 min) ligation of the LAD. Vehicle (saline, 0.5 μL/hr) or leptin (0.62 μg/day) was infused chronically for 28 days starting 20 min after reperfusion using osmotic minipumps connected to the ICV cannula. ECHO assessment of cardiac function was performed every week. At the end of week 4, +dP/dt max and Tau were accessed by LV catheterization. Hearts were then collected for evaluation of MT function in isolated cardiac fibers using Oroboros oxygraphy-2k respirometer. ICV leptin treatment improved systolic and diastolic function as evidenced by increased ejection fraction 4 weeks after I/R (46±3 vs. 26±3 %), stroke volume (353±19 vs. 193±27 μL), +dP/dt max (10387±1686 vs. 5022±442 mmHg/s) and reduced Tau (6.5±0.3 vs. 8±0.3 ms) when compared with vehicle-treated rats. In addition, ICV leptin infusion significantly increased ATP-linked respiration (55±3 vs. 39±1 %), reduced proton leak (45±3 vs. 61±1 %), and improved MT reserve capacity (42±5 vs. 27±2 %). Improved MT function was associated with increased MT Complex I- mediated respiration (47±6 vs. 26±1 pmolO 2 /s - mg). ICV leptin treatment also increased PGC1 α protein expression and COX3 gene expression, indicating enhanced MT biogenesis. These results demonstrate that chronic ICV leptin infusion improves cardiac function following I/R injury and suggests that leptin’s CNS-mediated cardioprotective effects may involve improved myocardial MT function and biogenesis.

Heart failure has a high mortality rate, and current therapies offer limited benefits. The authors demonstrate that activation of the central nervous system leptin-melanocortin pathway confers remarkable protection against progressive heart failure following severe myocardial infarction. The beneficial cardiac-protective actions of leptin require activation of brain melanocortin-4 receptors and elicit improvements in cardiac substrate oxidation, cardiomyocyte contractility, Ca2+ coupling, and mitochondrial efficiency. These findings highlight a potentially novel therapeutic approach for myocardial infarction and heart failure.

Background Obesity and hypertension are risk factors for myocardial infarction (MI); however, their potential interactions on post‐MI outcomes are unclear. We examined interactions of obesity and hypertensionon post‐MI function, remodeling, metabolic changes, and recovery. Methods and Results Male and female C57BL/6J mice were provided standard chow or high‐fat/fructose diet for 8 weeks and then infused with angiotensin II for 2 weeks to induce hypertension. MI was then induced by surgical ligation of the left coronary artery for 7 days. Obesity alone did not cause cardiac injury or exacerbate hypertension‐induced cardiac dysfunction. After MI, however, obese‐normotensive mice had lower survival rates compared with chow‐fed mice (56% versus 89% males; 54% versus 75% females), which were further decreased by hypertension (29% males; and 35% females). Surviving obese‐normotensive males displayed less left ventricular dilation and pulmonary congestion compared with chow‐fed males after MI; hypertension reversed left ventricular dilation because of high‐fat/fructose diet and promoted significant pulmonary congestion compared with chow‐fed controls. Obese‐normotensive males displayed higher left ventricular α‐MHC (alpha‐myosin heavy chain) protein, phosphorylated Akt (protein kinase B) and AMPK (adenosine‐monophosphate activated kinase), PPAR‐γ (peroxisome proliferator activated receptor gamma), and plasma adiponectin levels after MI, indicating favorable contractile and metabolic changes. However, these favorable contractile and metabolic changes were attenuated by hypertension. Obese‐hypertensive males also had lower levels of collagen in the infarcted region, indicating decreased ability to promote an adaptive wound healing response to MI. Conclusions Obesity reduces post‐MI survival but is associated with improved post‐MI cardiac function and metabolism in surviving normotensive mice. When hypertension accompanies obesity, favorable metabolic pathways associated with obesity are attenuated and post‐MI cardiac function and remodeling are adversely impacted.

Myocardial infarction (MI) is one of the leading causes of mortality and cardiovascular disease worldwide. MI is characterized by a substantial inflammatory response in the infarcted left ventricle (LV), followed by transition of quiescent fibroblasts to active myofibroblasts, which deposit collagen to form the reparative scar. Metabolic shifting between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) is an important mechanism by which these cell types transition towards reparative phenotypes. Thus, we hypothesized that dimethyl fumarate (DMF), a clinically approved anti-inflammatory agent with metabolic actions, would improve post-MI remodeling via modulation of macrophage and fibroblast metabolism. Adult male C57BL/6J mice were treated with DMF (10 mg/kg) for 3-7 days after MI. DMF attenuated LV infarct and non-infarct wall thinning at 3 and 7 days post-MI, and decreased LV dilation and pulmonary congestion at day 7. DMF improved LV infarct collagen deposition, myofibroblast activation, and angiogenesis at day 7. DMF also decreased pro-inflammatory cytokine expression (Tnf) 3 days after MI, and decreased inflammatory markers in macrophages isolated from the infarcted heart (Hif1a, Il1b). In fibroblasts extracted from the infarcted heart at day 3, RNA-Seq analysis demonstrated that DMF promoted an anti-inflammatory/pro-reparative phenotype. By Seahorse analysis, DMF did not affect glycolysis in either macrophages or fibroblasts at day 3, but enhanced macrophage OXPHOS while impairing fibroblast OXPHOS. Our results indicate that DMF differentially affects macrophage and fibroblast metabolism, and promotes anti-inflammatory/pro-reparative actions. In conclusion, targeting cellular metabolism in the infarcted heart may be a promising therapeutic strategy.

Introduction: Previous studies suggest that the perinatal environment can profoundly impact long-term metabolic health of the offspring. Hypothesis: We tested the hypothesis that parental (paternal + maternal) obesity impairs cardiac function in the offspring early in life. Methods: Within 1-3 days after weaning, offspring from obese rats fed a high fat diet (HFD-Offs) and age-matched offspring from lean rats (ND-Offs) were submitted to echocardiography and cardiac catheterization for assessment of pressure-volume relationships. Then, hearts were digested and isolated cardiomyocytes were used to determine contractile function, calcium transients, proteins related to calcium signaling, and mitochondrial bioenergetics. Results: Female and male HFD-Offs were heavier (72±2 and 61±4 vs 57±2 and 49 ±1 g), hyperglycemic (112±8 and 115±12 vs 92±10 and 96±8 mg/dL), with higher plasma insulin and leptin concentrations compared to female and male ND-Offs respectively. Compared to male ND-Offs controls, male HFD-Offs exhibited similar systolic function but impaired diastolic function as indicated by increased isovolumetric relaxation time (IVRT, 22±1 vs. 17±1 ms), the ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (E’) (E/E’ ratio, 29±2 vs. 23±1) and Tau (5.7±0.2 vs. 4.8±0.2 ms). The impaired diastolic function was associated with reduced resting free Ca 2+ levels and phospholamban protein expression, increased activated matrix metalloproteinase 2 and reduced sirtuin 3 (SIRT3) protein expression, mitochondrial ATP reserve and ATP-linked respiration. We found no differences in diastolic function or SIRT3 expression levels in female HFD-Offs and ND-Offs. Conclusions: These results indicate that male and female offspring from obese parents have metabolic abnormalities early in life (1-3 days after weaning) and that male, but not female, Offs from obese parents have impaired diastolic dysfunction and reductions in cardiac SIRT3, resting free Ca +2 levels and mitochondrial bioenergetics. (R01 DK121411, NHLBI-PO1HL51971, NIGMS P20GM104357 and U54GM115428)

We found that parental obesity leads to high blood pressure (BP) and diastolic dysfunction in male offspring. Here, we examined the impact of parental obesity on BP regulation, systolic and diastolic function, and exercise tolerance in female offspring. The following groups were studied: 1) offspring fed normal diet (N) born from N lean parents (N-F1-N, n=5); 2) offspring fed high fat diet (H) born from N parents (N-F1-H, n=6); 3) offspring fed N born from H obese parents (H-F1-N, n=7); and 4) offspring fed H born from H obese parents (H-F1-H, n=7). Parents were fed H or N from weaning until the end of lactation, while offspring were placed on N or H diet from weaning until 22 weeks of age. Body weight, fat and lean mass were measured weekly (EchoMRI4-in-1 System). Cardiac function including ejection fraction (EF), isovolumetric relaxation time (IVRT), early (E’) and late (A’) mitral annular diastolic velocities were measured by echocardiography (VisualSonics VEVO-3100) using left ventricle long and short axes, pulse wave and tissue Doppler. BP, measured by telemetry, was similar among all groups. Compared to lean N-F1-N control mice, H-F1-N mice had similar body weight (23.7±0.1 vs. 25.2±1.8 g), fat mass (3.4±0.1 vs. 4.4±1.6 g) and lean mass (22.9±0.4 vs. 24.6±0.3 g). Both offspring groups fed H after weaning were heavier than offspring fed normal diet, however, H-F1-H mice were significantly heavier than N-F1-H mice (41.2±1.6 vs. 37.4±1.0 g) due to greater fat mass (16.2±1.1 vs. 13.2±1.0 g) and lean mass (25.1±1.7 vs. 22.9 vs. 0.3 g). Despite no major differences in EF among all 4 groups, mice born from obese H parents exhibited signs of diastolic dysfunction, irrespective of their own diet, as indicated by reduced E’/A’ ratio (0.8±0.1 and 1.2±0.2 vs. 1.6±0.1 and 1.5±0.2, for H-F1-H and H-F1-N vs. N-F1-N and H-F1-H, respectively), reduced exercise capacity (4.2±0.7 and 4.7±0.5 vs. 5.9±0.7 and 7.4±0.3 m.kg), and increased IVRT in obese H-F1-H compared to obese N-F1-H (28.4±2.3 vs. 22.2±0.8 ms). These results indicate that despite no alterations in BP diet-induced parental obesity is associated with signs of diastolic dysfunction and impaired fitness in female offspring, which is exacerbated by continuing to feed the offspring with H diet.

While myocardial reperfusion is the most effective therapy to reduce mortality after myocardial infarction, it can paradoxically exacerbate ischemic injury. We recently showed that leptin, via its actions on the central nervous system (CNS), improved left ventricular function in a model of heart failure induced by permanent ligation of the left anterior coronary artery (LAD). To investigate whether leptin also protects the heart against ischemia/reperfusion (I/R) injury via its actions in the CNS, we instrumented female Wistar rats (15 weeks of age) with an intracerebroventricular (ICV) cannula into the lateral ventricle, and after 7 days of recovery and baseline assessment of cardiac function by echocardiography (VisualSonics VEVO-3100®), myocardial I/R was induced by temporary LAD ligation (60 min). Vehicle (saline, 0.5 μL/hr, n=6) or leptin (15 μg/day, n=7) were infused ICV for 28 consecutive days starting 20 min after reperfusion using osmotic minipump connected to the ICV cannula. Echocardiographic assessment of cardiac function was performed every week and at the end of the 4 th week of treatment, the heart was collected and processed for protein analysis and histological evaluation of infarct size and collagen deposition. Compared to vehicle treatment, chronic ICV leptin infusion significantly reduced infarcted area (21±2 vs. 37±4 %), septal collagen deposition (2.2±0.2 vs. 4.0±0.7 %), and markedly attenuated systolic dysfunction as evidenced by increased ejection fraction 4 weeks post I/R (59±1 vs. 30±2%), stroke volume (296±19 vs. 159±8 μL) and cardiac output (108±5 vs.63±4 μL/min). ICV leptin infusion also prevented the increase in left atrium to aorta diameter ratio (1.5±0.1 vs. 2±0.1 mm), an index of cardiac congestion. In addition, we found a 2-fold increase in the ratio of phospho-acetyl-CoA carboxylase (p-ACC) to total ACC protein expression, a marker of fatty acid oxidation, in hearts of leptin-treated rats compared with vehicle infusion. These results indicate that leptin exerts powerful beneficial CNS-mediated effects on the heart that improve systolic function and protect the myocardium against I/R injury. Our results also suggest that these beneficial actions may involve improved myocardial bioenergetics.

Transient receptor potential cation channel subfamily C member 6 (TPRC6) is a receptor‐operated cation channel that modulates cell Ca2+ influx. TRPC6 is expressed in many tissues including brain and adipose tissue. We previously showed that whole‐body TRPC6 deletion (TRPC6 KO) causes obesity, impaired glucose tolerance, and leptin resistance in mice fed a standard diet (SD). However, the metabolic consequences of feeding a high fat diet (HFD) to TRPC6 KO mice are still unknown. In this study, we investigated the impact of HFD (45% Cal from fat) from 6 to 18 weeks of age in male and female TRPC6 KO and B6/129s background control mice on body weight (BW), body fat/lean composition, food intake (FI), responses to acute leptin injection, and tolerance to an oral glucose load (OGTT). TRPC6 KO mice of both sexes were heavier than control mice at 6 weeks of age before starting HFD (27.3 ± 1.0 vs 24.2 ± 0.8 g in males and 24.1 ± 0.8 vs 19.1 ± 0.7 in females, n=7/sex, p<0.05). After 10 weeks on HFD, male and female TRPC6 KO mice gained less weight compared to control mice (BW gain: 8.5 ± 0.9 vs 15.2 ± 1.2 g in males and 9.4 ± 0.8 vs 12.2 ± 1.0 g in females from 6 to 16 weeks of age). EchoMRI scans showed that the percentage of lean mass was slightly lower while the percentage of fat mass was higher in TRPC6 KO mice compared to controls at the beginning of HFD. After 10 weeks on HFD, male and female TRPC6 KO mice gained less fat mass than control mice (fat mass gain: 4.2 ± 0.5 vs 11.6 ± 0.6 g in males and 5.7 ± 0.7 vs 8.8 ± 0.6 g in females from 6 to16 weeks of age), while lean mass increased similarly in both genotypes. Daily food intake in TRPC6 KO mice was significantly higher during the first 3 weeks of HDF (average daily FI: 3.5 ± 0.1 vs 2.8 ± 0.1 g in males, and 3.1 ± 0.1 vs 2.6 ± 0.1 in females from 6–8 weeks of age, n=7, p<0.05), after which FI was similar between groups (2.8 ± 0.1 vs 2.7 ± 0.1 g in males and 2.6 ± 0.1 vs 2.4 ± 0.1 in females from 9–16 weeks of age, n=7). After 12 weeks on a HFD, an acute leptin injection (5 mg/kg, i.p.) in fasted mice (n=7/sex/genotype) reduced 24‐hr FI in control mice by 0 % in males and 21% in females. In contrast, leptin reduced 24‐hr FI in TRPC6 KO mice by 15 % in males and 25% in females. An OGTT was performed after 12 weeks on a HFD and male and female TRPC6 KO mice exhibited impaired glucose tolerance compared to control mice (AUC: 19,198 ± 1,357 vs 9,450 ± 2,101 mg/dL × 120 min in males; and 17,117 ± 1,987 vs 5,470 ± 371 mg/dL × 120 min in females, n=7, p<0.05). Our results show that although TRPC6 KO mice have increased adiposity and BW on a SD, they appear to be resistant to HFD‐induced fat mass gain while exhibiting significant intolerance to glucose. Although TRPC6 may play an important role in the regulation of BW, FI, fat mass and glucose handling when fed a SD or HFD, the mechanisms involved in the different metabolic responses in RD and HFD are still unclear and may involve reduced sensitivity to leptin or differential roles of TRPC6 in brain.Support or Funding InformationNHLBI PO1HL51971, NIGMS P20GM104357 and U54GM115428, and NIDDK R00DK113280

Transient receptor potential cation channel C (TRPC) is essential for hypoxic pulmonary vasoconstriction response and alveolar gas exchange. We previously showed that whole body TRCP6 deficient (TRPC6 KO) mice are obese and resistant to the anorexic effects of leptin, a hormone that modulates ventilatory control. In the present study we investigated ventilatory responses to hypercapnia (7% CO2) in male and female 22‐week‐old TRPC6 KO and B6/129 control mice fed normal or high fat diet (HFD) since weaning (n=5 per group/sex/diet). Pulmonary ventilation (VE), tidal volume (VT) and respiratory frequency (FR) were measured using the plethysmography method. TRPC6 KO mice were heavier than control mice when fed normal diet (46.2±1.9 vs. 36.6±2.2 g in males and 34.0±1.4 vs. 26.9±1.3 g in females). However, when fed a HFD, male and female TRPC6 KO and B6/129 mice had similar body weight (43.6±0.4 vs. 47.5±1.4 g and 40.1±0.6 vs. 40.7±2.5 g, respectively). Compared to B6/129 controls male and female TRPC6 KO mice fed normal diet had similar VE (1817± 233 vs. 2139±135 and 2146± 404 vs. 2943±421 ml/min/kg) and VT (10.9±0.6 vs. 9.9±0.6 and 17.0±3.4 vs. 14.6±0.8 ml/kg), but lower FR (130±9 vs. 217±14 and 127±5 vs. 201±26 breaths/min) at room air. During hypercapnia, only male TRPC6 KO mice exhibited lower VE (3631± 493 vs. 6003±698 ml/min/kg), which was due to lower FR (235 ±16 vs. 305±19 breaths/min) and VT (15.3±1.0 vs. 19.5±1.4 ml/kg) responses to 7% CO2. Although female TRPC6 KO mice also showed lower FR to hypercapnia compared to B6/129 controls (242±6 vs. 313±22 breaths/min) they exhibited higher VT (28.4±3.0 vs. 25.8±2.0 ml/kg), thus resulting in similarly increased VE (6914±847 vs. 8119±956 ml/min/kg). Male and female B6/129 mice fed a HFD had significantly reduced VE responses to 7% CO2 (4020± 332 vs. 6003±698 and 5140± 194 vs. 8119±956 ml/min/kg, respectively) that were mainly caused by reduced VT (14.4±1.3 vs. 19.5±1.4 and 18.8±0.6 vs. 25.8±2.0 ml/kg). No further reduction in in ventilatory responses to hypercapnia were observed in male or female TPRC6 KO mice fed a HFD. These results suggest that TRPC6 plays an important role in ventilatory responses to hypercapnia, albeit more profound in males than females, and that HFD significantly reduces ventilatory responses to hypercapnia in both male and female control mice, but not in TRPC6 KO mice. The mechanisms involved in the reduced ventilatory responses are still unclear but may involve reduced sensitivity to leptin evoked by HFD or TRPC6 deficiency.Support or Funding InformationNIDDK 1RO1121411, NHLBI‐PO1HL51971, NIGMS P20GM104357 and U54GM115428

The proportion of women and men of reproductive age who are overweight and obese has been increasing considerably, and maternal obesity and excessive gestational weight gain may predispose their offspring to health‐related consequences during childhood and adulthood. Obesity may also be associated with increased cardiovascular responses to mental stress. However, there is no available evidence on the effects of intergenerational obesity on cardiovascular responses to acute stress in adult offspring. In this study, we examined whether the pressor response to acute stress is exacerbated in obese offspring from obese parents. Offspring fed normal diet (ND) born from ND parents (N‐F1‐N, n=5 per sex) used as control, offspring fed high fat diet (HFD) born from ND parents (N‐F1‐H, n=5 per sex) and offspring fed HFD born from HFD parents (H‐F1‐H, n=5 per sex) were used. Parents were fed HFD or ND after they were weaned, while the offspring were placed on HFD from weaning until the end of experiments. Mice were implanted with telemetry probes for measurement of mean arterial pressure (MAP) and heart rate (HR). After a 10 day recovery period, baseline MAP and HR were continuously measured for 30 minutes before, during 5‐minute air‐jet stress consisting of pulses of compressed air directed at the animal’s forehead, and 30 minutes after the stress test. Male and female H‐F1‐H mice were heavier than N‐F1‐H and N‐F1‐N control mice (47.3±0.8 vs. 42.5±1.9 and 33.1±0.2 g and 41.1±0.1 vs. 37.2±0.1 and 24.1±0.1 g, respectively). Male H‐F1‐H mice had higher MAP (110±4 mmHg) compared to N‐F1‐H and control mice (100±6 and 99±3 mmHg). Acute air‐jet stress significantly increased MAP by 36±2 mmHg in male H‐F1‐H mice compared to only 26±3 and 28±3 mmHg in male N‐F1‐H and control mice, respectively. MAP remained significantly higher post stress in H‐F1‐H mice (133±2 vs. 114±6 and 110±3 mmHg). Baseline HR and HR responses to air‐jet were similar among the male groups. In females, however, baseline MAP and HR as well as MAP and HR responses to air‐jet stress test were similar among groups. These results indicate that male, but not female, offspring fed a HFD from obese parents have higher baseline blood pressure (BP) and greater BP responses during acute stress, and their BP remains elevated for a longer period post stress. These observations also suggest that parental obesity may have greater effect on male offspring cardiovascular system compared to female offspring.Support or Funding InformationNIDDK 1RO1121411, NHLBI‐PO1HL51971, NIGMS P20GM104357 and U54GM115428

Obesity is strongly associated with cardiomyopathy and heart failure. We examined the impact of paternal and maternal obesity on systolic and diastolic function in male adult offspring. The following groups were studied: 1) offspring fed normal diet (ND) born from ND parents (ND-F1-ND, n=5); 2) offspring fed high fat diet (HFD) born from ND parents (HFD-F1-ND, n=6); 3) offspring fed ND born from HFD parents (ND-F1-HFD, n=7); and 4) offspring fed HFD born from HFD parents (HFD-F1-HFD, n=7). Parents were fed HFD or ND from weaning until the end of lactation, while the offspring were placed on ND or HFD from weaning until the end of experiments. Body weight, fat and lean mass, measured using EchoMRI4-in-1 System, were measured weekly. Cardiac function including ejection fraction (EF), isovolumetric relaxation time (IVRT), late filling velocity (A’), E and E’ early filling velocities were determined at 22 weeks of age using long and short axis left ventricle dimensions, pulse wave and tissue Doppler (VisualSonics 30 MHZ transducer, VEVO3100®). Compared to lean control ND-F1-ND mice, ND-F1-HFD mice had similar body weight (28.9±1.0 vs. 27.9±0.4 g) but higher fat mass (4.5±0.9 vs. 3.0±0.3 g) and lower lean mass (22.9±0.4 vs. 24.6±0.3 g). Both groups of mice fed HFD after weaning were heavier than offspring fed ND. However, HFD-F1-HFD mice were heavier than HFD-F1-ND mice (47.3±0.8 vs. 42.5±1.9 g) due to greater fat mass (19.4±0.6 vs. 15.7±1.6 g) and lean mass (28.3±0.6 vs. 26.9 vs. 0.3 g). Despite no major differences in ejection fraction among all 4 groups, mice fed HFD or born from parents fed HFD exhibited diastolic dysfunction; worse indicators of diastolic function were observed in mice from HFD parents as indicated by increased IVRT (30.4±1.8, 25.0±1.8, 22.1±2.0 vs. 21.4±0.9 ms) and E/E’ ratio (36.6±3.6, 32.3±1.8, 31.8±1.2 vs. 27.9±2.5) and reduced E’/A’ ratio (0.9±0.1, 0.7±0.1, 1.5±0.2 vs. 1.4±0.1) for HFD-F1-HFD, ND-F1-HFD, HFD-F1-ND and ND-F1-ND, respectively. These results indicate that diet-induced parental obesity is associated with diastolic dysfunction and preserved EF in the offspring, which is exacerbated by HFD-induced obesity. (NHLBI-PO1HL51971, NIGMS P20GM104357 and U54GM115428)

Transient receptor potential cation channel C (TRPC) is a group of receptor‐operated cation channels that modulate cell Ca2+ influx. TPRC6 is a subunit of the TRPC family that is expressed in the brain. However, the role of TRPC6 in controlling metabolic and cardiovascular functions remains unknown. In this study, we investigated the impact of genetic TRPC6 deletion on energy balance, metabolic and cardiovascular functions, and anorexic responses to leptin. Metabolic phenotypes including body weight (BW), food intake (FI), body fat/lean composition, energy expenditure (EE), respiratory quotient (RQ), responses to acute leptin injection as well as cardiovascular parameters including blood pressure (BP) and heart rate (HR) were measured by telemetry in male and female TRPC6 null and B6/129 control mice (n=7/per group and sex). TRPC6 null mice were heavier than control mice from 6 to 16 weeks of age when fed a standard diet (39.5±1.5 vs 31.1±1.2 g in males and 31.1±1.2 vs 22.9±1.0 g in females at 16 weeks of age, p<0.05). EchoMRI scans showed that the higher BW of TRPC6 null mice was mainly due to increased body fat compared to controls (30.0 vs 22.6 % of fat mass/BW in males and 32.7 vs 21.0 % in females at 16 weeks of age), and was associated with increased FI (3.4±0.1 vs 2.7±0.2 g/day in males and 2.7±0.1 vs 2.0±0.1 g/day in females at 16 weeks of age, p<0.05). EE, assessed by indirect calorimetry, was significantly reduced in TRPC6 null mice (0.108±0.003 vs 0.205±0.012 kcal/12h/g in males and 0.178±0.005 vs 0.217±0.006 kcal/12h/g in females at 17 weeks of age, p<0.05) and was accompanied by significantly reduced 24‐h average RQ (0.80±0.01 vs 0.89±0.01 in males and 0.77±0.01 vs 0.84± 0.01 in females at 17 weeks of age, p<0.05) when compared to male and female controls. Acute leptin injections (5 mg/kg, i.p. at 18 weeks of age) reduced 24‐hr FI by 41% and 32 % in male and female control mice, respectively, while only 8 % and 18 % reductions in FI were observed in male and female TRPC6 null mice. BP and HR measured by telemetry for 5 consecutive days at 22 weeks of age were similar in TRPC6 null mice and control mice (MAP: 111±2 vs 108±2 in males and 103±3 vs 108±3 in females; HR: 541±9 vs 526±14 in males and 552±4 vs 552±27 in females). Our results indicate that TRPC6 plays an important role in normal control of FI, BW and energy balance as well as for normal anorexic responses to leptin. Although TRPC6 deficiency caused obesity and metabolic abnormalities, BP and HR did not increase suggesting that TRPC6 may be an important link between obesity and hypertension.Support or Funding InformationNHLBI PO1HL51971, NIGMS P20GM104357 and U54GM115428, and NIDDK R00DK113280This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The brain leptin‐melanocortin system pathway plays an important role in regulating many physiological functions including energy homeostasis. We found that chronic intracerebroventricular (ICV) infusion of leptin or the melanocortin 4 receptor (MC4R) agonist Melanotan II, MTII, markedly attenuated cardiac dysfunction in rats with myocardial infarction (MI) induced by permanent left anterior descending coronary artery (LAD) ligation. In the present study we examined if activation of leptin‐MC4R axis in the central nervous system (CNS) alters cardiac substrate utilization in rats with MI. Male Sprague‐Dawley rats at 15 weeks of age were implanted with an ICV cannula into the lateral ventricle. After 10 days of recovery, the LAD was permanently ligated and saline vehicle (0.5 μL/hr, n=5), leptin (15 μg/day, n=5) or MTII (240 ng/day, n=3) was infused ICV via osmotic minipump for 14 consecutive days. On day 14 of treatment the hearts were quickly excised and perfused ex vivo in the working mode with glucose (5.5 mmol/L) and the fatty acid oleate (0.4 mmol/L). After a 5‐min baseline equilibration period the hearts were perfused for an additional 20 min and coronary flow samples were collected every 5 min to determine rates of substrate oxidation by quantitative measurement of [ 14 C]O 2 and [ 3 H] 2 O using [U‐ 14 C]glucose (0.08 μCi/ml) and [9,10‐ 3 H]oleate (0.1 μCi/ml) as the radiolabeled tracers. Compared to vehicle treatment, chronic ICV leptin infusion for 2 weeks markedly increased glucose oxidation (0.60±0.04 vs. 0.27±0.02 μmol/min/g dry heart weight) without altering oleate oxidation (0.95±0.01 vs. 0.90±0.02 μmol/min/g dry heart weight). Chronic central MTII infusion did not alter glucose oxidation (0.25±0.03 vs. 0.27±0.02 μmol/min/g dry heart weight) but increased fatty acid oxidation compared to vehicle treatment (1.13±0.01 vs. 0.90±0.02 μmol/min/g dry heart weight). These results suggest the CNSmechanisms by which leptin and MC4R activation improve cardiac function post MI may involve increased myocardial bioenergetics by differential modulation of glucose and fatty acid utilization. Support or Funding Information NHLBI PO1HL51971, NIGMS P20GM104357 and NIGMS U54GM115428 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .