Abstract Cardiac metabolism is highly adaptive in response to changes in substrate availability, as occur during fasting. This metabolic flexibility is essential to the maintenance of contractile function and is under the control of a group of select transcriptional regulators, notably the nuclear receptor family of factors member PPARα. However, the diversity of physiologic and pathologic states through which the heart must sustain function suggests the possible existence of additional transcriptional regulators that play a role in matching cardiac metabolism to energetic demand. Here we show that cardiac KLF15 is required for the normal cardiac response to fasting. Specifically, we find that cardiac function is impaired upon fasting in systemic and cardiac specific Klf15-null mice. Further, cardiac specific Klf15-null mice display a fasting-dependent accumulation of long chain acylcarnitine species along with a decrease in expression of the carnitine translocase Slc25a20. Treatment with a diet high in short chain fatty acids relieves the KLF15-dependent long chain acylcarnitine accumulation and impaired cardiac function in response to fasting. Our observations establish KLF15 as a critical mediator of the cardiac adaptive response to fasting through its regulation of myocardial lipid utilization.

Citation: Sugi K, Hsieh PN, Ilkayeva O, Shelkay S, Moroney B, Baadh P, et al. (2018) Kruppel-like factor 15 is required for the cardiac adaptive response to fasting. PLoS ONE 13(2): e0192376. https://doi.org/10.1371/journal.pone.0192376 Editor: Elena Aikawa, Brigham and Women's Hospital, Harvard Medical School, UNITED STATES Received: October 10, 2017; Accepted: January 20, 2018; Published: February 6, 2018 Copyright: © 2018 Sugi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper. Funding: This work was supported, in whole or in part, by National Institutes of Health Grants F32HL110538 and K01DK104922 (to D.A.P.), R01HL112486, R01HL119195, and R01HL123098 (to M.J.K.), F30AG054237 (to P.N.H), along with generous gifts by Tom F. Peterson (TFP Lab). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The adult heart sustains contractile function regardless of fuel supply, in the face of enormous metabolic demand. Indeed, from an ATP pool of approximately 5 μmol/g wet weight, the heart uses 0.5 μmol/g wet weight per second and therefore perpetually verges on ATP depletion [1]. Although the heart demonstrates a preference for the oxidation of fatty acids, as approximately 70% of the ATP generated in the myocardium is derived from them, ~20% comes from glucose and lactate, and the remainder from diverse substrates (e.g. ketones) [2]. Furthermore, the heart is endowed with the capacity to adapt substrate utilization to match the systemic environment. During fasting, the myocardium increases its dependence on oxidative catabolism of fatty acids and inhibits glycolysis, events which occur simultaneously with a rise in expression of genes involved in fatty acid oxidation [3, 4]. The importance of this adaptive capacity to enhance lipid utilization is illustrated by the clinical features of patients with inborn errors of myocardial fatty acid oxidation, where episodic manifestations of the disease often occur following fasting, presumably as the heart relies more heavily on fatty acid oxidation to meet its energetic demands. [5] Myocardial lipid utilization is a complex multi-step process [2]. Fatty acids enter the myocardium via passive diffusion or facilitated by carriers including fatty acid translocase (FAT)/CD36, the plasma membrane isoform of fatty acid binding protein (FABPpm), or fatty acid transport protein (FATP) 1/6, with CD36 accounting for 50–60% of fatty acid oxidation [6]. Long chain cytosolic fatty acids are esterified by fatty acyl CoA synthetase (FACS) and, if destined for oxidation, further transferred to carnitine via the mitochondrial enzyme carnitine palmitoyltransferase 1 (CPT1) and shuttled across the outer mitochondrial membrane. Translocation of acylcarnitines across the inner mitochondrial membrane is accomplished by a carnitine:acylcarnitine translocase (Slc25a20), moving the fatty acid moiety into the mitochondrial matrix, and simultaneously generating free carnitine. Carnitine palmitoyltransferase 2 (CPT2) subsequently converts long-chain acylcarnitines into acyl CoA species, which are free to enter the β-oxidation pathway. Fasting induced alterations in myocardial lipid utilization are achieved, in part, through the activity of several key transcriptional regulators. Work performed by others over the last decade has identified roles for nuclear receptors including the peroxisome proliferator-activated receptors (PPARs), estrogen-related receptors (ERRs), and nuclear respiratory factors 1 and 2 (NRF-1 and -2) in the regulation of genes, among others, involved in cardiac fatty acid oxidation [7]. Recently, we have provided evidence that a zinc-finger DNA-binding transcription factor, Kruppel-like factor 15 (KLF15), is also a critical regulator of myocardial substrate metabolism, especially lipid utilization, and cooperates with PPARα to coordinately regulate gene expression [8, 9]. Further, KLF15 is required for PPARα mediated induction of canonical fasting-inducible gene targets [8, 9]. Given our observations, we therefore postulated that KLF15 regulation of cardiac metabolism might be necessary for metabolic adaptation to substrate availability, as occurs under fasting regimens. Here, we demonstrate that Klf15-null hearts exhibit impaired function during fasting, coincident with an accumulation of long chain acyl-carnitines and decrease in transcript levels of Slc25a20. These phenotypes are rescued by a diet high in short chain fatty acids, which do not require transport into the mitochondria. Together, our findings suggest KLF15 is critical to the cardiac fasting response and point to a model whereby KLF15 regulation of Slc25a20 during the fasting state enables transport of long chain acylcarnitine species into mitochondria for oxidation.

Materials and methods Animal models Animal protocols were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University. Protocols were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Klf15-/- and cardiac-specific KLF15-cKO mice have been previously described [10, 11]. Male, age-matched controls on a C57Bl/6 genetic background were used. Mice were maintained with ad libitum access to water and standard laboratory chow (Laboratory diet P3000; 4.5% fat by weight, 14% kilocalories). Barrier facility had a 12 hour light/dark cycle and was temperature and humidity controlled. For fasting experiments, food was removed at indicated times and lasted for up to 48 hours in duration. Where indicated, mice were fed a short chain fatty acid enriched diet (SCD: Harlan Teklad TD.09849) for 10-weeks as previously described [12]. Plasma parameters and tissue lipids Hearts and plasma were rapidly harvested from WT and KO mice fed ad libitum or fasted for two days. Lipids were extracted from ventricles by the Folch method. Cardiac fatty acid and triglyceride content was quantified by thin-layer chromatography by the NIH-MMPC / Lipid Analytic Core at Vanderbilt University. Plasma free-fatty acids and triglycerides were quantified by the Vanderbilt NIH Mouse Metabolic Phenotyping Core. Plasma glucose measurements were performed using the glucose colorimetric / fluorometric assay kit from BioVision according to the manufacturer’s instructions. Acylcarnitine measurements Hearts were rapidly harvested from WT and KO mice fed ad libitum or fasted for two days. Tissue homogenates (50 mg/mL) were prepared using a polytron in 50% acetonitrile and 0.3% formic acid. 45 acylcarnitine species (including short, medium, and long-chain) were analyzed by tandem mass spectrometry (MS/MS) using sample preparation methods described previously [13, 14]. The data were acquired using a Waters triple quadrupole detector equipped with AcquityTM UPLC system and controlled by MassLynx 4.1 software platform (Waters, Milford, MA). Echocardiographic analysis Transthoracic echocardiography was performed in anesthetized mice using the Vevo 770 High Resolution Imaging System (Visual Sonics) and the RMV-707B 30-MHz probe as previously described [10]. Cardiac function was assessed at the mid papillary muscle level in M-mode short axis images. RNA extraction and QPCR Heart tissue samples were disrupted in PureZOLTM (Biorad) in a Tissue-lyzer (Qiagen) using stainless steel beads (30 Hz for a total of 4 min). AurumTM (Biorad) RNA isolation kit was used to isolate total RNA according to manufacturer’s directions. For qPCR analysis, total RNA was loaded onto columns and DNase treated before being transcribed to complementary DNA using iScriptTM (Biorad) according to manufacturer’s directions. TaqMan method was used for qPCR analysis (using the Roche Universal Probe Library System) using an ABI Step One Plus Real-Time PCR System. ΔΔCt method (with normalization genes indicated in figure legend)was used to assess relative expression. Primer sequence for Slc25a20: Forward 5’-ATCCGCGGCTTCTACAAAG-3’, Reverse 5’-TACATCCCACTGGCAGGAAC-3’. Primer sequence for Cpt1b: Forward 5’-TGCCTTTACATCGTCTCCAA-3’, Reverse 5’-GGCTCCAGGGTTCAGAAAGT-3’. Primer sequence for Cpt2: Forward 5’-CCAAAGAAGCAGCGATGG-3’, Reverse 5’-TAGAGCTCAGGCAGGGTGA-3’. Primer sequence for Fabp3: Forward 5’-CTTTGTCGGTACCTGGAAGC-3’, Reverse 5’-TGGTCATGCTAGCCACCTG-3’. Primer sequence for Glut4: Forward 5’-TCGTCATTGGCATTCTGGT-3’, Reverse 5’-AGCAGTGGCCACAGGGTA-3’. Primer sequence for Glut1: Forward 5’-ATGGATCCCAGCAGCAAG-3’, Reverse 5’-CCAGTGTTATAGCCGAACTGC-3’. Primer sequence for Ppib: Forward 5’-TTCTTCATAACCACAGTCAAGACC-3’, Reverse 5’-ACCTCCGTACCACATCCAT-3’. Western blot analysis Protein was extracted and homogenized from heart tissue samples in RIPA buffer (Sigma-Aldrich, R0278) containing proteinase/phosphatase inhibitor cocktail (Roche). Samples in SDS sample buffer were run on SDS-PAGE and immunoblotted with anti-CACT (Sigma, 1:1000) and anti-α-tubulin (Cell Signaling, 1:2000). Secondary HRP-conjugated antibodies were from Cell Signaling and chemiluminescent detection reagent was from Thermo Scientific. Bio-Rad Quantity One software was used for quantitation. Two-tailed Student's t-test for unpaired data was used Data analysis All error bars depict SEM, and data is expressed as means. In experiments comparing the means of two normally distributed groups, two-tailed Student's t-test for unpaired data was used. In experiments comparing the means of normally distributed groups with multiple treatments, one-way analysis of variance (ANOVA) with the Tukey post hoc test was used. Statistical significance defined as p < 0.05.

Discussion Cardiac metabolic plasticity during fasting, namely the augmentation of lipid utilization, is crucial for its function [2, 19]. Transcriptional regulators coordinate this augmentation, and we establish here that KLF15 is critically required for cardiac lipid flux and function during fasting. In systemic and cardiac specific Klf15-null mice, we observe decreased fractional shortening under fasting conditions. Additionally, metabolomics analysis reveals an accumulation of long chain acylcarnitine species in hearts of cardiac specific Klf15-null mice during fasting, suggesting an inability of these hearts to adequately manage lipid flux during a nutrient deprived state. This theory is bolstered by our finding that a diet rich in short chain fatty acids, which cross easily into the mitochondria and therefore can act as an alternative energy source, can rescue both the long chain acyl-carnitine abnormalities as well as cardiac fractional shortening. Finally, we find by qPCR analysis that transcript levels of a key transporter of acyl-carnitines, Slc25a20, are lowered in cardiac specific Klf15-null mice hearts. Together with prior work, these results implicate cardiac KLF15 in the fasting response through its regulation of lipid flux and illustrate the significance of transcriptional control of cardiac metabolism in governing cardiac function. While coordinated alterations in cardiac metabolism are critical to the normal fasting response, metabolic derangements have also been shown to contribute to various disease states. In diabetic hearts, glucose oxidation is inhibited and there is decreased activation of PI3K-Akt signaling in response to insulin, while fatty acid oxidation is enhanced through increased circulating levels of free fatty acids as well as the aberrant action of PPARα, changes contributing to the development of a diabetes associated cardiomyopathy [20, 21]. Interestingly, the overexpression of PPARα in cardiomyocytes alone is sufficient to phenocopy many of the features of this cardiomyopathy, while PPARα deficiency is protective against a streptozotocin-induced model of diabetic cardiomyopathy [22, 23]. Given our prior description of a KLF15-PPARα molecular module regulating fatty acid oxidation, KLF15 may also play a role in diabetes-induced cardiac dysfunction [9]. Additionally, alterations in substrate usage and their attendant consequences have also been noted in the failing heart, namely a relative, although not absolute, increase in glucose oxidation and decrease in fatty acid oxidation, particularly in late stages of disease [24–28]. Notably, although fatty acid oxidation is reduced, lipid uptake into the cytosol is in fact enhanced, resulting in an accumulation of triglycerides in the cardiomyocyte as well as shunting of lipids into maladaptive non-oxidative pathways, broadly termed lipotoxicity [29, 30]. Previous work from our group has shown that mRNA transcripts of cardiac KLF15 are lowered in rodent and human samples of heart failure, and that the mouse with global loss of KLF15 is more susceptible to heart failure following pressure overload, suggesting a role for KLF15 regulation of fatty acid oxidation in the context of heart failure [8, 16, 31, 32]. The selective accumulation of unoxidized long chain acylcarnitines in the cardiac KLF15 knockout mouse, its exacerbation upon fasting, and its rescue with a diet of short chain fatty acids is reminiscent of the clinical phenotype of patients affected by genetic defects in long chain fatty acid oxidation pathways. These patients, who can harbor mutations in genes of fatty acid transport across the mitochondrial membrane or in specific enzymes of the β-oxidation pathway, exhibit metabolic decompensation during fasting which is alleviated with a diet enriched in medium chain fatty acids and depleted in long chain fatty acids [33–37]. The cardiac sequelae of an inborn error of fatty acid oxidation have been theoretically attributed to toxicity of the long chain acylcarnitine, either through the generation of reactive oxygen species, or through incorporation in the sarcolemma leading to generation of arrhythmias due to its amphiphilic nature; these mechanisms may also be operative in the KLF15 deficient mouse [38]. Indeed, in the diabetic cardiomyopathy model mouse overexpressing PPARα in the heart, Finck et al. observe an accumulation of triacylglycerides containing long chain fatty acids which is rescued upon treatment with a medium chain fatty acid diet [23]. Since, in our model, the relief of long chain acylcarnitine burden occurs simultaneously with the return of normal cardiac function, it seems likely that this accumulation of long chain acylcarnitines is also key to the deterioration in cardiac function of KLF15 knockout mice. Additionally, it remains unclear what mechanisms mediate the decrease in long chain acylcarnitine accumulation in the presence of an abundant alternative lipid energy source such as short chain fatty acids. However, as diets rich in short to medium chain fatty acids have been shown to be useful in some diseases of inborn defects of fatty acid oxidation, it will be valuable to consider their therapeutic value in other diseases in which fatty acid oxidation pathways are dysregulated, including diabetes and advanced heart failure.

Acknowledgments This work was supported, in whole or in part, by National Institutes of Health Grants F32HL110538 and K01DK104922 (to D.A.P.), R01HL112486, R01HL119195, and R01HL123098 (to M.J.K.), F30AG054237 (to P.N.H), along with generous gifts by Tom F. Peterson (TFP Lab).