Metabolic signatures of pregnancy-induced cardiac growth.

The goal of this study was to develop an atlas of the metabolic, transcriptional, and proteomic changes that occur with pregnancy in the maternal heart. Timed pregnancy studies in FVB/NJ mice revealed a significant increase in heart size by day 8 of pregnancy (midpregnancy; MP), which was sustained throughout the rest of the term compared with nonpregnant control mice. Cardiac hypertrophy and myocyte cross-sectional area were highest 7 days after birth (postbirth; PB) and were associated with significant increases in end-diastolic and end-systolic left ventricular volumes and higher cardiac output. Metabolomics analyses revealed that by day 16 of pregnancy (late pregnancy; LP) metabolites associated with nitric oxide production as well as acylcholines, sphingomyelins, and fatty acid species were elevated, which coincided with a lower activation state of phosphofructokinase and higher levels of pyruvate dehydrogenase kinase 4 (Pdk4) and ß-hydroxybutyrate dehydrogenase 1 (Bdh1). In the postpartum period, urea cycle metabolites, polyamines, and phospholipid levels were markedly elevated in the maternal heart. Cardiac transcriptomics in LP revealed significant increases in not only Pdk4 and Bdh1 but also genes that regulate glutamate and ketone body oxidation, which were preceded in MP by higher expression of transcripts controlling cell proliferation and angiogenesis. Proteomics analysis of the maternal heart in LP and PB revealed significant reductions in several contractile filament and mitochondrial subunit complex proteins. Collectively, these findings describe the coordinated molecular changes that occur in the maternal heart during and after pregnancy.NEW & NOTEWORTHY Little is known of the underlying molecular and cellular mechanisms that contribute to pregnancy-induced cardiac growth. Several lines of evidence suggest that changes in cardiac metabolism may contribute. Here, we provide a comprehensive metabolic atlas of the metabolomic, proteomic, and transcriptomic changes occurring in the maternal heart. We show that pregnancy-induced cardiac growth is associated with changes in glycerophospholipid, nucleotide, and amino acid metabolism, with reductions in cardiac glucose catabolism. Collectively, these results suggest that substantial metabolic changes occur in the maternal heart during and after pregnancy.

Cardiac PANK1 deletion exacerbates ventricular dysfunction during pressure overload.

Coenzyme A (CoA) is an essential cofactor required for intermediary metabolism. Perturbations in homeostasis of CoA have been implicated in various pathologies; however, whether CoA homeostasis is changed and the extent to which CoA levels contribute to ventricular function and remodeling during pressure overload has not been explored. In this study, we sought to assess changes in CoA biosynthetic pathway during pressure overload and determine the impact of limiting CoA on cardiac function. We limited cardiac CoA levels by deleting the rate-limiting enzyme in CoA biosynthesis, pantothenate kinase 1 (Pank1). We found that constitutive, cardiomyocyte-specific Pank1 deletion (cmPank1-/-) significantly reduced PANK1 mRNA, PANK1 protein, and CoA levels compared with Pank1-sufficient littermates (cmPank1+/+) but exerted no obvious deleterious impact on the mice at baseline. We then subjected both groups of mice to pressure overload-induced heart failure. Interestingly, there was more ventricular dilation in cmPank1-/- during the pressure overload. To explore potential mechanisms contributing to this phenotype, we performed transcriptomic profiling, which suggested a role for Pank1 in regulating fibrotic and metabolic processes during the pressure overload. Indeed, Pank1 deletion exacerbated cardiac fibrosis following pressure overload. Because we were interested in the possibility of early metabolic impacts in response to pressure overload, we performed untargeted metabolomics, which indicated significant changes to metabolites involved in fatty acid and ketone metabolism, among other pathways. Collectively, our study underscores the role of elevated CoA levels in supporting fatty acid and ketone body oxidation, which may be more important than CoA-driven, enzyme-independent acetylation in the failing heart.NEW & NOTEWORTHY Changes in CoA homeostasis have been implicated in a variety of metabolic diseases; however, the extent to which changes in CoA homeostasis impacts remodeling has not been explored. We show that limiting cardiac CoA levels via PANK deletion exacerbated ventricular remodeling during pressure overload. Our results suggest that metabolic alterations, rather than structural alterations, associated with Pank1 deletion may underlie the exacerbated cardiac phenotype during pressure overload.

Physiological Biomimetic Culture System for Pig and Human Heart Slices.

RATIONALE: Preclinical testing of cardiotoxicity and efficacy of novel heart failure therapies faces a major limitation: the lack of an in situ culture system that emulates the complexity of human heart tissue and maintains viability and functionality for a prolonged time. OBJECTIVE: To develop a reliable, easily reproducible, medium-throughput method to culture pig and human heart slices under physiological conditions for a prolonged period of time. METHODS AND RESULTS: Here, we describe a novel, medium-throughput biomimetic culture system that maintains viability and functionality of human and pig heart slices (300 µm thickness) for 6 days in culture. We optimized the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to ß-adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices. Furthermore, this culture system enabled the assessment of contraction and relaxation kinetics on isolated single myofibrils from heart slices after culture. CONCLUSIONS: We have developed and optimized a reliable medium-throughput culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics and reliable testing of cardiotoxicity in a 3-dimensional heart model.

Cardiac mesenchymal cells from failing and nonfailing hearts limit ventricular dilation when administered late after infarction.

Although cell therapy-mediated cardiac repair offers promise for treatment/management of heart failure, lack of fundamental understanding of how cell therapy works limits its translational potential. In particular, whether reparative cells from failing hearts differ from cells derived from nonfailing hearts remains unexplored. Here, we assessed differences between cardiac mesenchymal cells (CMC) derived from failing (HF) versus nonfailing (Sham) hearts and whether the source of donor cells (i.e., from HF vs. Sham) limits reparative capacity, particularly when administered late after infarction. To determine the impact of the donor source of CMCs, we characterized the transcriptional profile of CMCs isolated from sham (Sham-CMC) and failing (HF-CMC) hearts. RNA-seq analysis revealed unique transcriptional signatures in Sham-CMC and HF-CMC, suggesting that the donor source impacts CMC. To determine whether the donor source affects reparative potential, C57BL6/J female mice were subjected to 60 min of regional myocardial ischemia and then reperfused for 35 days. In a randomized, controlled, and blinded fashion, vehicle, HF-CMC, or Sham-CMC were injected into the lumen of the left ventricle at 35 days post-MI. An additional 5 weeks later, cardiac function was assessed by echocardiography, which indicated that delayed administration of Sham-CMC and HF-CMC attenuated ventricular dilation. We also determined whether Sham-CMC and HF-CMC treatments affected ventricular histopathology. Our data indicate that the donor source (nonfailing vs. failing hearts) affects certain aspects of CMC, and these insights may have implications for future studies. Our data indicate that delayed administration of CMC limits ventricular dilation and that the source of CMC may influence their reparative actions.NEW & NOTEWORTHY Most preclinical studies have used only cells from healthy, nonfailing hearts. Whether donor condition (i.e., heart failure) impacts cells used for cell therapy is not known. We directly tested whether donor condition impacted the reparative effects of cardiac mesenchymal cells in a chronic model of myocardial infarction. Although cells from failing hearts differed in multiple aspects, they retained the potential to limit ventricular remodeling.

Metabolic regulation of Kv channels and cardiac repolarization by Kvß2 subunits.

Voltage-gated potassium (Kv) channels control myocardial repolarization. Pore-forming Kvα proteins associate with intracellular Kvß subunits, which bind pyridine nucleotides with high affinity and differentially regulate channel trafficking, plasmalemmal localization and gating properties. Nevertheless, it is unclear how Kvß subunits regulate myocardial K+ currents and repolarization. Here, we tested the hypothesis that Kvß2 subunits regulate the expression of myocardial Kv channels and confer redox sensitivity to Kv current and cardiac repolarization. Co-immunoprecipitation and in situ proximity ligation showed that in cardiac myocytes, Kvß2 interacts with Kv1.4, Kv1.5, Kv4.2, and Kv4.3. Cardiac myocytes from mice lacking Kcnab2 (Kvß2-/-) had smaller cross sectional areas, reduced sarcolemmal abundance of Kvα binding partners, reduced Ito, IK,slow1, and IK,slow2 densities, and prolonged action potential duration compared with myocytes from wild type mice. These differences in Kvß2-/- mice were associated with greater P wave duration and QT interval in electrocardiograms, and lower ejection fraction, fractional shortening, and left ventricular mass in echocardiographic and morphological assessments. Direct intracellular dialysis with a high NAD(P)H:NAD(P)+ accelerated Kv inactivation in wild type, but not Kvß2-/- myocytes. Furthermore, elevated extracellular levels of lactate increased [NADH]i and prolonged action potential duration in wild type cardiac myocytes and perfused wild type, but not Kvß2-/-, hearts. Taken together, these results suggest that Kvß2 regulates myocardial electrical activity by supporting the functional expression of proteins that generate Ito and IK,slow, and imparting redox and metabolic sensitivity to Kv channels, thereby coupling cardiac repolarization to myocyte metabolism.

E2f1 deletion attenuates infarct-induced ventricular remodeling without affecting O-GlcNAcylation.

Several post-translational modifications figure prominently in ventricular remodeling. The beta-O-linkage of N-acetylglucosamine (O-GlcNAc) to proteins has emerged as an important signal in the cardiovascular system. Although there are limited insights about the regulation of the biosynthetic pathway that gives rise to the O-GlcNAc post-translational modification, much remains to be elucidated regarding the enzymes, such as O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which regulate the presence/absence of O-GlcNAcylation. Recently, we showed that the transcription factor, E2F1, could negatively regulate OGT and OGA expression in vitro. The present study sought to determine whether E2f1 deletion would improve post-infarct ventricular function by de-repressing expression of OGT and OGA. Male and female mice were subjected to non-reperfused myocardial infarction (MI) and followed for 1 or 4 week. MI significantly increased E2F1 expression. Deletion of E2f1 alone was not sufficient to alter OGT or OGA expression in a naïve setting. Cardiac dysfunction was significantly attenuated at 1-week post-MI in E2f1-ablated mice. During chronic heart failure, E2f1 deletion also attenuated cardiac dysfunction. Despite the improvement in function, OGT and OGA expression was not normalized and protein O-GlcNAcyltion was not changed at 1-week post-MI. OGA expression was significantly upregulated at 4-week post-MI but overall protein O-GlcNAcylation was not changed. As an alternative explanation, we also performed guided transcriptional profiling of predicted targets of E2F1, which indicated potential differences in cardiac metabolism, angiogenesis, and apoptosis. E2f1 ablation increased heart size and preserved remote zone capillary density at 1-week post-MI. During chronic heart failure, cardiomyocytes in the remote zone of E2f1-deleted hearts were larger than wildtype. These data indicate that, overall, E2f1 exerts a deleterious effect on ventricular remodeling. Thus, E2f1 deletion improves ventricular remodeling with limited impact on enzymes regulating O-GlcNAcylation.

Deficiency of aldose reductase exacerbates early pressure overload-induced cardiac dysfunction and autophagy in mice.

Pathological cardiac hypertrophy is associated with the accumulation of lipid peroxidation-derived aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and acrolein in the heart. These aldehydes are metabolized via several pathways, of which aldose reductase (AR) represents a broad-specificity route for their elimination. We tested the hypothesis that by preventing aldehyde removal, AR deficiency accentuates the pathological effects of transverse aortic constriction (TAC). We found that the levels of AR in the heart were increased in mice subjected to TAC for 2 weeks. In comparison with wild-type (WT), AR-null mice showed lower ejection fraction, which was exacerbated 2 weeks after TAC. Levels of atrial natriuretic peptide and myosin heavy chain were higher in AR-null than in WT TAC hearts. Deficiency of AR decreased urinary levels of the acrolein metabolite, 3-hydroxypropylmercapturic acid. Deletion of AR did not affect the levels of the other aldehyde-metabolizing enzyme - aldehyde dehydrogenase 2 in the heart, or its urinary product - (N-Acetyl-S-(2-carboxyethyl)-l-cystiene). AR-null hearts subjected to TAC showed increased accumulation of HNE- and acrolein-modified proteins, as well as increased AMPK phosphorylation and autophagy. Superfusion with HNE led to a greater increase in p62, LC3II formation, and GFP-LC3-II punctae formation in AR-null than WT cardiac myocytes. Pharmacological inactivation of JNK decreased HNE-induced autophagy in AR-null cardiac myocytes. Collectively, these results suggest that during hypertrophy the accumulation of lipid peroxidation derived aldehydes promotes pathological remodeling via excessive autophagy, and that metabolic detoxification of these aldehydes by AR may be essential for maintaining cardiac function during early stages of pressure overload.

Exercise-Induced Changes in Glucose Metabolism Promote Physiological Cardiac Growth.

BACKGROUND: Exercise promotes metabolic remodeling in the heart, which is associated with physiological cardiac growth; however, it is not known whether or how physical activity-induced changes in cardiac metabolism cause myocardial remodeling. In this study, we tested whether exercise-mediated changes in cardiomyocyte glucose metabolism are important for physiological cardiac growth. METHODS: We used radiometric, immunologic, metabolomic, and biochemical assays to measure changes in myocardial glucose metabolism in mice subjected to acute and chronic treadmill exercise. To assess the relevance of changes in glycolytic activity, we determined how cardiac-specific expression of mutant forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase affect cardiac structure, function, metabolism, and gene programs relevant to cardiac remodeling. Metabolomic and transcriptomic screenings were used to identify metabolic pathways and gene sets regulated by glycolytic activity in the heart. RESULTS: Exercise acutely decreased glucose utilization via glycolysis by modulating circulating substrates and reducing phosphofructokinase activity; however, in the recovered state following exercise adaptation, there was an increase in myocardial phosphofructokinase activity and glycolysis. In mice, cardiac-specific expression of a kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transgene (GlycoLo mice) lowered glycolytic rate and regulated the expression of genes known to promote cardiac growth. Hearts of GlycoLo mice had larger myocytes, enhanced cardiac function, and higher capillary-to-myocyte ratios. Expression of phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in the heart (GlycoHi mice) increased glucose utilization and promoted a more pathological form of hypertrophy devoid of transcriptional activation of the physiological cardiac growth program. Modulation of phosphofructokinase activity was sufficient to regulate the glucose-fatty acid cycle in the heart; however, metabolic inflexibility caused by invariantly low or high phosphofructokinase activity caused modest mitochondrial damage. Transcriptomic analyses showed that glycolysis regulates the expression of key genes involved in cardiac metabolism and remodeling. CONCLUSIONS: Exercise-induced decreases in glycolytic activity stimulate physiological cardiac remodeling, and metabolic flexibility is important for maintaining mitochondrial health in the heart.

Cardiomyocyte Ogt limits ventricular dysfunction in mice following pressure overload without affecting hypertrophy.

The myocardial response to pressure overload involves coordination of multiple transcriptional, posttranscriptional, and metabolic cues. The previous studies show that one such metabolic cue, O-GlcNAc, is elevated in the pressure-overloaded heart, and the increase in O-GlcNAcylation is required for cardiomyocyte hypertrophy in vitro. Yet, it is not clear whether and how O-GlcNAcylation participates in the hypertrophic response in vivo. Here, we addressed this question using patient samples and a preclinical model of heart failure. Protein O-GlcNAcylation levels were increased in myocardial tissue from heart failure patients compared with normal patients. To test the role of OGT in the heart, we subjected cardiomyocyte-specific, inducibly deficient Ogt (i-cmOgt -/-) mice and Ogt competent littermate wild-type (WT) mice to transverse aortic constriction. Deletion of cardiomyocyte Ogt significantly decreased O-GlcNAcylation and exacerbated ventricular dysfunction, without producing widespread changes in metabolic transcripts. Although some changes in hypertrophic and fibrotic signaling were noted, there were no histological differences in hypertrophy or fibrosis. We next determined whether significant differences were present in i-cmOgt -/- cardiomyocytes from surgically naïve mice. Interestingly, markers of cardiomyocyte dedifferentiation were elevated in Ogt-deficient cardiomyocytes. Although no significant differences in cardiac dysfunction were apparent after recombination, it is possible that such changes in dedifferentiation markers could reflect a larger phenotypic shift within the Ogt-deficient cardiomyocytes. We conclude that cardiomyocyte Ogt is not required for cardiomyocyte hypertrophy in vivo; however, loss of Ogt may exert subtle phenotypic differences in cardiomyocytes that sensitize the heart to pressure overload-induced ventricular dysfunction.

MicroRNA-539 is up-regulated in failing heart, and suppresses O-GlcNAcase expression.

Derangements in metabolism and related signaling pathways characterize the failing heart. One such signal, O-linked ß-N-acetylglucosamine (O-GlcNAc), is an essential post-translational modification regulated by two enzymes, O-GlcNAc transferase and O-GlcNAcase (OGA), which modulate the function of many nuclear and cytoplasmic proteins. We recently reported reduced OGA expression in the failing heart, which is consistent with the pro-adaptive role of increased O-GlcNAcylation during heart failure; however, molecular mechanisms regulating these enzymes during heart failure remain unknown. Using miRNA microarray analysis, we observed acute and chronic changes in expression of several miRNAs. Here, we focused on miR-539 because it was predicted to target OGA mRNA. Indeed, co-transfection of the OGA-3'UTR containing reporter plasmid and miR-539 overexpression plasmid significantly reduced reporter activity. Overexpression of miR-539 in neonatal rat cardiomyocytes significantly suppressed OGA expression and consequently increased O-GlcNAcylation; conversely, the miR-539 inhibitor rescued OGA protein expression and restored O-GlcNAcylation. In conclusion, this work identifies the first target of miR-539 in the heart and the first miRNA that regulates OGA. Manipulation of miR-539 may represent a novel therapeutic target in the treatment of heart failure and other metabolic diseases.

Induction of activating transcription factor 3 limits survival following infarct-induced heart failure in mice.

Numerous fibrotic and inflammatory changes occur in the failing heart. Recent evidence indicates that certain transcription factors, such as activating transcription factor 3 (ATF3), are activated during heart failure. Because ATF3 may be upregulated in the failing heart and affect inflammation, we focused on the potential role of ATF3 on postinfarct heart failure. We subjected anesthetized, wild-type mice to nonreperfused myocardial infarction and observed a significant induction in ATF3 expression and nuclear translocation. To test whether the induction of ATF3 affected the severity of heart failure, we subjected wild-type and ATF3-null mice to nonreperfused infarct-induced heart failure. There were no differences in cardiac function between the two genotypes, except at the 2-wk time point; however, ATF3-null mice survived the heart failure protocol at a significantly higher rate than the wild-type mice. Similar to the slight favorable improvements in chamber dimensions at 2 wk, we also observed greater cardiomyocyte hypertrophy and more fibrosis in the noninfarcted regions of the ATF3-null hearts compared with the wild-type. Nevertheless, there were no significant group differences at 4 wk. Furthermore, we found no significant differences in markers of inflammation between the wild-type and ATF3-null hearts. Our data suggest that ATF3 suppresses fibrosis early but not late during infarct-induced heart failure. Although ATF3 deficiency was associated with more fibrosis, this did not occur at the expense of survival, which was higher in the ATF3-null mice. Overall, ATF3 may serve a largely maladaptive role during heart failure.

Complement component 3 is necessary to preserve myocardium and myocardial function in chronic myocardial infarction.

Activation of the complement cascade (CC) with myocardial infarction (MI) acutely initiates immune cell infiltration, membrane attack complex formation on injured myocytes, and exacerbates myocardial injury. Recent studies implicate the CC in mobilization of stem/progenitor cells and tissue regeneration. Its role in chronic MI is unknown. Here, we consider complement component C3, in the chronic response to MI. C3 knockout (KO) mice were studied after permanent coronary artery ligation. C3 deficiency exacerbated myocardial dysfunction 28 days after MI compared to WT with further impaired systolic function and LV dilation despite similar infarct size 24 hours post-MI. Morphometric analysis 28 days post-MI showed C3 KO mice had more scar tissue with less viable myocardium within the infarct zone which correlated with decreased c-kit(pos) cardiac stem/progenitor cells (CPSC), decreased proliferating Ki67(pos) CSPCs and decreased formation of new BrdU(pos) /α-sarcomeric actin(pos) myocytes, and increased apoptosis compared to WT. Decreased CSPCs and increased apoptosis were evident 7 days post-MI in C3 KO hearts. The inflammatory response with MI was attenuated in the C3 KO and was accompanied by attenuated hematopoietic, pluripotent, and cardiac stem/progenitor cell mobilization into the peripheral blood 72 hours post-MI. These results are the first to demonstrate that CC, through C3, contributes to myocardial preservation and regeneration in response to chronic MI. Responses in the C3 KO infer that C3 activation in response to MI expands the resident CSPC population, increases new myocyte formation, increases and preserves myocardium, inflammatory response, and bone marrow stem/progenitor cell mobilization to preserve myocardial function.

Cardiomyocyte Ogt is essential for postnatal viability.

The singly coded gene O-linked-ß-N-acetylglucosamine (O-GlcNAc) transferase (Ogt) resides on the X chromosome and is necessary for embryonic stem cell viability during embryogenesis. In mature cells, this enzyme catalyzes the posttranslational modification known as O-GlcNAc to various cellular proteins. Several groups, including our own, have shown that acute increases in protein O-GlcNAcylation are cardioprotective both in vitro and in vivo. Yet, little is known about how OGT affects cardiac function because total body knockout (KO) animals are not viable. Presently, we sought to establish the potential involvement of cardiomyocyte Ogt in cardiac maturation. Initially, we characterized a constitutive cardiomyocyte-specific (cm)OGT KO (c-cmOGT KO) mouse and found that only 12% of the c-cmOGT KO mice survived to weaning age (4 wk old); the surviving animals were smaller than their wild-type littermates, had dilated hearts, and showed overt signs of heart failure. Dysfunctional c-cmOGT KO hearts were more fibrotic, apoptotic, and hypertrophic. Several glycolytic genes were also upregulated; however, there were no gross changes in mitochondrial O2 consumption. Histopathology of the KO hearts indicated the potential involvement of endoplasmic reticulum stress, directing us to evaluate expression of 78-kDa glucose-regulated protein and protein disulfide isomerase, which were elevated. Additional groups of mice were subjected to inducible deletion of cmOGT, which did not produce overt dysfunction within the first couple of weeks of deletion. Yet, long-term loss (via inducible deletion) of cmOGT produced gradual and progressive cardiomyopathy. Thus, cardiomyocyte Ogt is necessary for maturation of the mammalian heart, and inducible deletion of cmOGT in the adult mouse produces progressive ventricular dysfunction.

Kv beta complex facilitates exercise-induced augmentation of myocardial perfusion and cardiac growth.

The oxygen sensitivity of voltage-gated potassium (Kv) channels regulates cardiovascular physiology. Members of the Kv1 family interact with intracellular Kvß proteins, which exhibit aldo-keto reductase (AKR) activity and confer redox sensitivity to Kv channel gating. The Kvß proteins contribute to vasoregulation by controlling outward K+ currents in smooth muscle upon changes in tissue oxygen consumption and demand. Considering exercise as a primary physiological stimulus of heightened oxygen demand, the current study tested the role of Kvß proteins in exercise performance, exercise-induced adaptations in myocardial perfusion, and physiological cardiac growth. Our findings reveal that genetic ablation of Kvß2 proteins diminishes baseline exercise capacity in mice and attenuates the enhancement in exercise performance observed after long-term training. Moreover, we demonstrate that Kvß2 proteins are critical for exercise-mediated enhancement in myocardial perfusion during cardiac stress as well as adaptive changes in cardiac structure. Our results underscore the importance of Kvß proteins in metabolic vasoregulation, highlighting their role in modulating both exercise capacity and cardiovascular benefits associated with training. Furthermore, our study sheds light on a novel molecular target for enhancing exercise performance and improving the health benefits associated with exercise training in patients with limited capacity for physical activity.

Dietary Branched Chain Amino Acids Modify Post-Infarct Cardiac Remodeling and Function in the Murine Heart.

Introduction: Branch-chain amino acids (BCAA) are markedly elevated in the heart following myocardial infarction (MI) in both humans and animal models. Nevertheless, it remains unclear whether dietary BCAA levels influence post-MI remodeling. We hypothesize that lowering dietary BCAA levels prevents adverse cardiac remodeling after MI. Methods and Results: To assess whether altering dietary BCAA levels would impact circulating BCAA concentrations, mice were fed a low (1/3×), normal (1×), or high (2×) BCAA diet over a 7-day period. We found that mice fed the low BCAA diet had >2-fold lower circulating BCAA concentrations when compared with normal and high BCAA diet feeding strategies; notably, the high BCAA diet did not further increase BCAA levels over the normal chow diet. To investigate the impact of dietary BCAAs on cardiac remodeling and function after MI, male and female mice were fed either the low or high BCAA diet for 2 wk prior to MI and for 4 wk after MI. Although body weights or heart masses were not different in female mice fed the custom diets, male mice fed the high BCAA diet had significantly higher body and heart masses than those on the low BCAA diet. Echocardiographic assessments revealed that the low BCAA diet preserved stroke volume and cardiac output for the duration of the study, while the high BCAA diet led to progressive decreases in cardiac function. Although no discernible differences in cardiac fibrosis, scar collagen topography, or cardiomyocyte cross-sectional area were found between the dietary groups, male mice fed the high BCAA diet showed longer cardiomyocytes and higher capillary density compared with the low BCAA group. Conclusions: Provision of a diet low in BCAAs to mice mitigates eccentric cardiomyocyte remodeling and loss of cardiac function after MI, with dietary effects more prominent in males.

Metabolic Pathways for Removing Reactive Aldehydes are Diminished in Atrophic Muscle During Heart Failure.

Background: Muscle wasting is a serious complication in heart failure patients, and oxidative stress is involved in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE) and acrolein, which causemuscle wasting. In tissues, these toxic aldehydes are metabolically removed by enzymes such asaldo keto reductases and endogenous nucleophiles, such as glutathione and carnosine. Whether these metabolic pathways could be affected in skeletal muscle during heart failure has never been studied. Methods: Male wild-type C57BL/6J mice were subjected to a pressure overload model of hypertrophy by transaortic constriction (TAC) surgery, and echocardiography was performed after 14 weeks. Different skeletal muscle beds were weighed and analyzed for atrophic and inflammatory markers, Atrogin1 and TRIM63, TNF-α and IL-6, respectively, by RT-PCR. Levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, aldose reductase (AKR1B1) and aldehyde dehydrogenase 2 (ALDH2) were measured by Western blotting, and histidyl dipeptides and histidyl dipeptide aldehyde conjugates were analyzed by LC/MS-MS in the gastrocnemius and soleus muscles of sham- and TAC-operated mice. Furthermore, histidyl dipeptide synthesizing enzyme carnosine synthase (CARNS) and amino acid transporters (PEPT2 and TAUT)wasmeasured in the gastrocnemius muscles of the sham and TAC-operated mice. Results: TAC-induced heart failure decreases body weight and gastrocnemius and soleus muscle weights. The expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, wasincreased (~1.5-2-fold), and the formation of HNE and acrolein-protein adducts was increased in the gastrocnemius muscle of TAC-operated mice. The expression of AKR1B1 remained unchanged, whereas ALDH2 was decreased, in the gastrocnemius muscle of TAC mice. Similarly, in the atrophic gastrocnemius muscle, levels of total histidyl dipeptides (carnosine and anserine) and, in particular,carnosine were decreased. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, the expression of CARNS and TAUT wasdecreased in the atrophic gastrocnemius muscle. Conclusions: Collectively, these results show that metabolic pathways involved in the removal of lipid peroxidation products and synthesis of histidyl dipeptides are diminished in atrophic skeletal muscle during heart failure, which could contribute to muscle atrophy.

Monocarboxylate transporter 2 and stroke severity in a rodent model of sleep apnea.

Stroke is not only more prevalent but is also associated with more severe adverse functional outcomes among patients with sleep apnea. Monocarboxylate transporters (MCT) are important regulators of cellular bioenergetics, have been implicated in brain susceptibility to acute severe hypoxia (ASH), and could underlie the unfavorable prognosis of cerebrovascular accidents in sleep apnea patients. Rodents were exposed to either intermittent hypoxia (IH) during sleep, a characteristic feature of sleep apnea, or to sustained hypoxia (SH), and expression of MCT1 and MCT2 was assessed. In addition, the functional recovery to middle cerebral artery occlusion (MCAO) in rats and hMCT2 transgenic mice and of hippocampal slices subjected to ASH was assessed, as well as the effects of MCT blocker and MCT2 antisense oligonucleotides and siRNAs. IH, but not SH, induced significant reductions in MCT2 expression over time at both the mRNA and protein levels and in the functional recovery of hippocampal slices subjected to ASH. Similarly, MCAO-induced infarcts were significantly greater in IH-exposed rats and mice, and overexpression of hMCT2 in mice markedly attenuated the adverse effects of IH. Exogenous pyruvate treatment reduced infarct volumes in normoxic rats but not in IH-exposed rats. Administration of the MCT2 blocker 4CN, but not the MCT1 antagonist p-chloromercuribenzene sulfonate, increased infarct size. Thus, prolonged exposures to IH mimicking sleep apnea are associated with increased CNS vulnerability to ischemia that is mediated, at least in part, by concomitant decreases in the expression and function of MCT2. Efforts to develop agonists of MCT2 should provide opportunities to ameliorate the overall outcome of stroke.

Direct measurement of blood flow in microvessels grown in Matrigel in vivo.

BACKGROUND: The Matrigel assay provides a versatile platform to examine vessel growth. Similarly, the microsphere method is used extensively in laboratory animals to measure tissue-specific blood flow. However, microsphere models have not been used with Matrigel to study angiogenesis in live animals. The goal of this study was to develop a novel technique to directly measure blood flow with microspheres in vessels grown in Matrigel in vivo. METHODS: In calves (n = 10, 110 ± 5 kg), 5 mL of Matrigel was injected subcutaneously. After 10 d, a percutaneous cardiac catheterization was performed. Fluorescent-labeled 15 µm microspheres were injected into the left ventricular chamber to distribute throughout the body based on systemic blood flow patterns. Afterwards, Matrigel plugs were removed, and animals were recovered. Flow cytometry was used to count microspheres and quantify blood flow within the plug. FITC-conjugated isolectin-B4 staining was performed to quantify Matrigel capillary density. Flow cytometry was performed to quantify circulating plasma CD34(+) cells. Linear regressions were used to determine relationships between Matrigel blood flow, Matrigel capillary density, and plasma CD34(+) cells. RESULTS: Over 10 d, small-caliber vessels grew into subcutaneous Matrigel plugs. Microspheres lodged throughout the plug and indicated that newly grown vessels in the Matrigel were functional and able to accommodate blood flow. Modest associations between Matrigel blood flow, Matrigel capillary density, and circulating plasma CD34(+) cells were noted. CONCLUSION: This method provides a novel and cost-effective technique to measure blood flow within vessels grown in Matrigel in vivo.

Collagen type XIX regulates cardiac extracellular matrix structure and ventricular function.

The cardiac extracellular matrix plays essential roles in homeostasis and injury responses. Although the role of fibrillar collagens have been thoroughly documented, the functions of non-fibrillar collagen members remain underexplored. These include a distinct group of non-fibrillar collagens, termed, fibril-associated collagens with interrupted triple helices (FACITs). Recent reports of collagen type XIX (encoded by Col19a1) expression in adult heart and evidence of its enhanced expression in cardiac ischemia suggest important functions for this FACIT in cardiac ECM structure and function. Here, we examined the cellular source of collagen XIX in the adult murine heart and evaluated its involvement in ECM structure and ventricular function. Immunodetection of collagen XIX in fractionated cardiovascular cell lineages revealed fibroblasts and smooth muscle cells as the primary sources of collagen XIX in the heart. Based on echocardiographic and histologic analyses, Col19a1 null (Col19a1N/N) mice exhibited reduced systolic function, thinning of left ventricular walls, and increased cardiomyocyte cross-sectional areas-without gross changes in myocardial collagen content or basement membrane morphology. Col19a1N/N cardiac fibroblasts had augmented expression of several enzymes involved in the synthesis and stability of fibrillar collagens, including PLOD1 and LOX. Furthermore, second harmonic generation-imaged ECM derived from Col19a1N/N cardiac fibroblasts, and transmission electron micrographs of decellularized hearts from Col19a1N/N null animals, showed marked reductions in fibrillar collagen structural organization. Col19a1N/N mice also displayed enhanced phosphorylation of focal adhesion kinase (FAK), signifying de-repression of the FAK pathway-a critical mediator of cardiomyocyte hypertrophy. Collectively, we show that collagen XIX, which had a heretofore unknown role in the mammalian heart, participates in the regulation of cardiac structure and function-potentially through modulation of ECM fibrillar collagen structural organization. Further, these data suggest that this FACIT may modify ECM superstructure via acting at the level of the fibroblast to regulate their expression of collagen synthetic and stabilization enzymes.

Bovine model of doxorubicin-induced cardiomyopathy.

Left ventricular assist devices (LVADs) constitute a recent advance in heart failure (HF) therapeutics. As the rigorous experimental assessment of LVADs in HF requires large animal models, our objective was to develop a bovine model of cardiomyopathy. Male calves (n = 8) were used. Four animals received 1.2 mg/kg intravenous doxorubicin weekly for seven weeks and four separate animals were studied as controls. Doxorubicin-treated animals were followed with weekly echocardiography. Target LV dysfunction was defined as an ejection fraction ≤ 35%. Sixty days after initiating doxorubicin, a terminal study was performed to determine hemodynamic, histological, biochemical, and molecular parameters. All four doxorubicin-treated animals exhibited significant (P < 0.05) contractile dysfunction, with target LV dysfunction achieved in three animals. Doxorubicin-treated hearts exhibited significantly reduced coronary blood flow and interstitial fibrosis and significantly increased apoptosis and myocyte size. Gene expression of atrial natriuretic factor increased more than 3-fold. Plasma norepinephrine and epinephrine levels were significantly increased early and late during the development of cardiomyopathy, respectively. We conclude that sequential administration of intravenous doxorubicin in calves induces a cardiomyopathy with many phenotypic hallmarks of the failing human heart. This clinically-relevant model may be useful for testing pathophysiologic responses to LVADs in the context of HF.