Diabetes is associated with an increased incidence of heart failure with preserved ejection fraction (HFpEF), but the underlying mechanisms are poorly understood. A shortage of mouse models reflecting the diverse HFpEF pathophysiology contributes to this inadequate understanding of disease mechanisms. We conducted a comprehensive analysis of a nongenetic, inducible type 2 diabetes mellitus (T2DM) mouse model about its suitability as a preclinical model of cardiometabolic, diabetes-induced HFpEF. T2DM was induced in C57Bl/6 mice by a high-fat/high-sucrose diet and a low-dose streptozotocin (DIO-STZ). Cardiac function was assessed in vivo by echocardiography and left ventricular catheterization and in vitro using the isolated perfused heart. Structural, molecular, and bioenergetic disturbances were analyzed by immunohistochemistry, RNA-seq, qPCR, Western blot, and extracellular flux analysis of myocardial tissue. Blood glucose, fatty acids, and ketone body levels were elevated, and insulin levels were reduced in DIO-STZ compared with chow. DIO-STZ mice showed an HFpEF phenotype with reduced cardiac output, end-diastolic volume, and increased filling pressure. No differences in myocardial fibrosis or in vitro stiffness were detected between DIO-STZ and chow. RNA-Seq pointed toward disturbances in lipid and ketone metabolism. Extracellular flux analysis revealed increased fatty acid oxidation capacity without differences in glucose metabolism. No general mitochondrial dysfunction was observed, but a reduced capacity for β-hydroxybutyrate oxidation. The diabetic DIO-STZ mouse model showed a pronounced functional HFpEF phenotype with underlying mechanisms that remarkably differ from other HFpEF models, making the DIO-STZ model a relevant extension of the range of HFpEF mouse models, especially for investigating molecular mechanisms or therapeutic interventions in diabetes-associated HFpEF.NEW & NOTEWORTHY Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome whose pathophysiological mechanisms are incompletely understood, potentially due to a lack of preclinical models reflecting the broad range of pathophysiological aspects. We describe a diabetic DIO-STZ mouse model showing a pronounced HFpEF with underlying mechanisms that remarkably differ from other HFpEF models, making this model a relevant extension of the range of HFpEF models, especially for investigating molecular mechanisms or therapeutical interventions in diabetes.

Background Diabetes and obesity are associated with an increased incidence of heart failure with preserved ejection fraction (HFpEF), but the underlying pathophysiological mechanisms are poorly understood. A shortage of appropriate preclinical mouse models reflecting different pathophysiological disease aspects might contribute to this inadequate understanding of the complex and diverse HFpEF pathophysiology. We conducted a comprehensive analysis of a non-genetic, inducible T2DM mouse model with regard to its suitability as a preclinical model of cardiometabolic, diabetes-induced HFpEF. Methods T2DM was induced in C57Bl/6 mice by high-fat/high-sucrose diet (HFHSD) combined with low-dose streptozotocin (STZ) treatment (DIO-STZ), control animals received standard chow (chow). As additional control groups, animals of a DIO group were solely fed HFHSD throughout the study, and a STZ group received solely STZ injections while maintained on a standard chow. Cardiac function was assessed in vivo by echocardiography and left ventricular catheterization, as well as in vitro using the isolated perfused heart model. Structural, molecular and bioenergetic disturbances were analyzed by immunohistochemistry, RNA-seq, qPCR, western blot, and extracellular flux analysis of myocardial tissue, among others. Results Blood glucose, fatty acids and ketone body levels were elevated, and insulin plasma level were reduced in DIO-STZ animals compared to chow. DIO-STZ mice showed a strong cardiometabolic HFpEF phenotype with reduced cardiac output, end-diastolic volume, and increased filling pressure. Neither STZ nor DIO mice showed signs of HFpEF development. No difference in myocardial fibrosis nor in in vitro myocardial stiffness was detected between DIO-STZ and chow. Myocardial RNA-Seq clearly pointed towards disturbances in lipid and ketone metabolism. Extracellular flux analysis in intact cardiac tissue slices revealed an increased fatty acid oxidation capacity without differences in glucose metabolism. Mitochondrial respirometry revealed no indication of general mitochondrial dysfunction or mitochondrial uncoupling, but a reduced capacity for β-hydroxybutyrate oxidation. Conclusions The diabetic DIO-STZ mouse model showed a pronounced functional HFpEF phenotype. However, we show clear evidence that the underlying mechanism differs remarkably from the other HFpEF models making the DIO-STZ model a relevant extension of the range of HFpEF mouse models, especially for investigating molecular mechanisms or therapeutical interventions in diabetes associated HFpEF. ### Competing Interest Statement The authors have declared no competing interest. * HFpEF : Heart failure with preserved ejection fraction HFrEF : Heart failure with reduced ejection fraction T2DM : Type 2 diabetes mellitus SGLT2i : Sodium-glucose transport inhibitor GLP-1RA : Glucagon-like peptide-1 receptor agonist HFHSD : High-fat and high-succrose diet HFD : High-fat diet DIO : Diet-induced obesity STZ : Streptozotocin NEFA : Non-esterified fatty acid ECG : Electrocardiogram PSLAX : Parasternal long axis SAX : Short axis EDV : End-diastolic volume ESV : End-systolic volume LVPW : Left ventricular posterior wall LVID : Left ventricular internal diameter RWT : Relative wall thickness GLS : Global longitudinal strain GLSR : Global longitudinal strain rate LV : Left ventricle AOPmean : Mean aortic pressure LVEDP : Left ventricular end-diastolic pressure LVPmin : Minimal left ventricular pressure LVPmax : Maximal left ventricular pressure DGE : Differentially expressed gene BDH1 : Beta-hydroxybutyrate dehydrogenase PDK4 : Pyruvate dehydrogenase kinase 4 OXCT1 : 3-oxoacid CoA-transferase 1 SCOT : Succinyl-CoA:3-ketoacid-CoA transferase CPT1 : Carnitine palmitoyltransferase 1 CPT2 : Carnitine palmitoyltransferase 2 PLIN2 : Perilipin-2 PLIN5 : Perilipin-5 HMGCS2 : 3-hydroxy-3-methylglutaryl-CoA synthase 2 RCR : Respiratory contral ratio NNT : Nicotinamide nucleotide transhydrogenase dP/dtmax : Maximal speed of left ventricular pressure rise dP/dtmin : Maximal speed of left ventricular pressure decay OCR : Oxygen consumption rate Deutsche Forschungsgemeinschaft, 236177352-CRC1116; projects A06, S01 Ministerium fM-CM-<r Kultur und Wissenschaft des Landes Nordrhein-Westfalen, https://ror.org/04n00c532, MODS; project 5 Forschungskommission der Medizinischen Fakultaet der Heinrich-Heine-Universitaet Duesseldorf, Doctoral scholarship to L.B. and Z.F.

AIM:Cardiac pathologies are accompanied by alterations in substrate metabolism, and extracellular flux analysis is a standard tool to investigate metabolic disturbances, especially in immortalized cell lines. However, preparations of primary cells, such as adult cardiomyocytes require enzymatic dissociation and cultivation affecting metabolism. Therefore, we developed a flux analyzer-based method for the assessment of substrate metabolism in intact vibratome-sliced mouse heart tissue.METHODS:Oxygen consumption rates were determined using a Seahorse XFe24-analyzer and "islet capture plates." We demonstrate that tissue slices are suitable for extracellular flux analysis and metabolize both free fatty acids (FFA) and glucose/glutamine. Functional integrity of tissue slices was proven by optical mapping-based assessment of action potentials. In a proof-of-principle approach, the sensitivity of the method was tested by analyzing substrate metabolism in the remote myocardium after myocardial infarction (I/R).RESULTS:Here, I/R increased uncoupled OCR compared with sham animals indicating a stimulated metabolic capacity. This increase was caused by a higher glucose/glutamine metabolism, whereas FFA oxidation was unchanged.CONCLUSION:In conclusion, we describe a novel method to analyze cardiac substrate metabolism in intact cardiac tissue slices by extracellular flux analysis. The proof-of-principle experiment demonstrated that this approach has a sensitivity allowing the investigation of pathophysiologically relevant disturbances in cardiac substrate metabolism.

The progression of cardiac diseases is often accompanied by disturbances in substrate metabolism. Extracellular flux analysis has become a standard tool to investigate metabolic alterations in cell lines. However, the enzymatic digestion of the heart to isolate adult cardiomyocytes as well as the cultivation procedure that is required for cell attachment to the cell culture plates might affect metabolism. Therefore, we developed a flux analyser-based method to measure substrate metabolism of intact cardiac tissue slices. Furthermore, we tested this method in a proof-of-principle approach in remote myocardium after myocardial infarction. To yield cardiac tissue pieces of comparable size, mouse cardiac tissue was sliced (150 µm) using a vibratome, and tissue pieces (diameter 1.9 mm) of these slices were punched out. Using "islet capture plates" in a Seahorse XFe 24 analyser, oxygen consumption rates (OCR) were measured at baseline and after FCCP-induced uncoupling in palmitate, glucose (Glc) and glutamine (Gln) enriched medium. To determine long-chain fatty acid metabolism, CPT1 was inhibited by etomoxir, and Glc/Gln metabolism by inhibition of mitochondrial pyruvate carrier (MPC) and glutaminase (Gls) with UK5099/BPTES. Optical mapping of membrane potential was used to assess action potentials in tissue slices as indicator of cellular integrity. Finally, the developed method was used to analyse substrate metabolism in the remote myocardium at day 3 after myocardial ischemia and reperfusion (n=7) in comparison to sham mice (n=8). Data are mean±SD; unpaired two-sample t-test. Basal OCR was 53±8 pmol/min, and FCCP increased OCR to 92±18 pmol/min. Both etomoxir and UK5099/BPTES reduced OCR indicating that both palmitate and Glc/Gln are metabolised. Optical mapping of tissue slices showed regular action potential characteristics and propagation. After myocardial infarction, CPT1 inhibition caused a smaller reduction of uncoupled mitochondrial OCR in I/R animals compared to sham (40±13 vs. 52±4%, p<0.05). This effect was caused by an increased metabolism of Glc/Glu (37±13 vs. 24±4 pmol/min, p<0.05), whilst the effect of CPT1 was not different. Here, we describe a new method to analyse cardiac metabolism using cardiac tissue slices that metabolise fatty acids as well as glucose, and show high functional integrity. Therefore, this method has the potential to expand the methodological alternatives to investigate cardiac substrate metabolism. In a proof-of-principle approach, the analysis of cardiac substrate metabolism of the remote myocardium after I/R showed an augmented glucose/glutamine metabolism.

Background Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) acts as a transcriptional coactivator and regulates mitochondrial function. Various isoforms are generated by alternative splicing and differentially regulated promoters. In the heart, total PGC-1α deficiency knockout leads to dilatative cardiomyopathy, but knowledge on the complexity of cardiac isoform expression of PGC-1α remains sparse. Thus, this study aims to generate a reliable dataset on cardiac isoform expression pattern by long-read mRNA sequencing, followed by investigation of differential regulation of PGC-1α isoforms under metabolic and ischemic stress, using high-fat-high-sucrose-diet-induced obesity and a murine model of myocardial infarction. Results Murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery) was used for long-read mRNA sequencing, resulting in full-length transcriptomes including 58,000 mRNA isoforms with 99% sequence accuracy. Automatic bioinformatic analysis as well as manual similarity search against exonic sequences leads to identification of putative coding PGC-1α isoforms, validated by PCR and Sanger sequencing. Thereby, 12 novel transcripts generated by hitherto unknown splicing events were detected. In addition, we postulate a novel promoter with homologous and strongly conserved sequence in human heart. High-fat diet as well as ischemia/reperfusion (I/R) injury transiently reduced cardiac expression of PGC-1α isoforms, with the most pronounced effect in the infarcted area. Recovery of PGC-1α-isoform expression was even more decelerated when I/R was performed in diet-induced obese mice. Conclusions We deciphered for the first time a complete full-length transcriptome of the murine and human heart, identifying novel putative PGC-1α coding transcripts including a novel promoter. These transcripts are differentially regulated in I/R and obesity suggesting transcriptional regulation and alternative splicing that may modulate PGC-1α function in the injured and metabolically challenged heart.

Acute myocardial infarction (MI) induces an extensive sterile inflammation, which is dominated in the early phase by invading neutrophils and monocytes/macrophages. The inflammatory response after MI critically affects infarct healing and cardiac remodeling. Therefore, modulation of cardiac inflammation may improve outcome post MI. Insulin-like growth factor 1 (IGF1) treatment reduces infarct size and improves cardiac function after MI via IGF1 receptor mediated signaling in myeloid cells. Our study aimed to investigate the effect of IGF1 on neutrophil phenotype both in vitro and in vivo after MI. We show that IGF1 induces an anti-inflammatory phenotype in bone marrow derived neutrophils. On the molecular and functional level IGF1 treated neutrophils were indistinguishable from those induced by IL4. Surprisingly, insulin, even though it is highly similar to IGF1 did not create anti-inflammatory neutrophils. Notably, the IGF1 effect was independent of the canonical Ras/Raf/ERK or PI3K/AKT pathway, but depended on activation of the JAK2/STAT6 pathway, which was not activated by insulin treatment. Single cell sequencing analysis 3 days after MI also showed that 3 day IGF1 treatment caused a downregulation of pro-inflammatory genes and upstream regulators in most neutrophil and many macrophage cell clusters whereas anti-inflammatory genes and upstream regulators were upregulated. Thus, IGF1 acts like an anti-inflammatory cytokine on myeloid cells in vitro and attenuates the pro-inflammatory phenotype of neutrophils and macrophages in vivo after MI. IGF1 treatment might therefore represent an effective immune modulatory therapy to improve the outcome after MI.

Conditional, cell-type-specific transgenic mouse lines are of high value in cardiovascular research. A standard tool for cardiomyocyte-restricted DNA editing is the αMHC-MerCreMer/loxP system. However, there is an ongoing debate on the occurrence of cardiac side effects caused by unspecific Cre activity or related to tamoxifen/oil overload. Here, we investigated potential adverse effects of DNA editing by the αMHC-MerCreMer/loxP system in combination with a low-dose treatment protocol with the tamoxifen metabolite 4-hydroxytamoxifen (OH-Txf). αMHC-MerCreMer mice received intraperitoneally OH-Txf (20 mg/kg) for 5 or 10 days. These treatment protocols were highly efficient to induce DNA editing in adult mouse hearts. Multi-parametric magnetic resonance imaging revealed neither transient nor permanent effects on cardiac function during or up to 19 days after 5 day OH-Txf treatment. Furthermore, OH-Txf did not affect cardiac phosphocreatine/ATP ratios assessed by in vivo 31P MR spectroscopy, indicating no Cre-mediated side effects on cardiac energy status. No MRI-based indication for the development of cardiac fibrosis was found as mean T1 relaxation time was unchanged. Histological analysis of myocardial collagen III content after OH-Txf confirmed this result. Last, mean T2 relaxation time was not altered after Txf treatment suggesting no pronounced cardiac lipid accumulation or tissue oedema. In additional experiments, cardiac function was assessed for up to 42 days to investigate potential delayed side effects of OH-Txf treatment. Neither 5- nor 10-day treatment resulted in a depression of cardiac function. Efficient cardiomyocyte-restricted DNA editing that is free of unwanted side effects on cardiac function, energetics or fibrosis can be achieved in adult mice when the αMHC-MerCreMer/loxP system is activated by the tamoxifen metabolite OH-Txf.

Initially, the function of the fat mass and obesity associated (Fto) gene seemed to be primarily the regulation of the body weight. Here we show that loss of Fto results in a hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis. In consequence, Fto(-/-) mice display an anxiety-like behavior and impairments in working memory. Furthermore, differentiation of neurons is affected in the hippocampus. As a cause of these impairments we identified a processing defect of the neurotrophin BDNF which is most likely the result of a reduced expression of MMP-9. Therefore, we propose FTO as a possible new target to develop novel approaches for the treatment of diseases associated with hippocampal disorders. In parallel, we also would like to make the point that any anti-obesity therapy via blocking FTO function can have negative effects on the proper function of the hippocampus.

Insulin‐like growth factor (IGF1) controls growth and metabolism of many cell types. In addition, IGF1 has been shown to provide cardioprotective effects after acute myocardial infarction (AMI). However, the cell type and mechanisms involved are not yet fully understood. Mice were exposed to 45 min LAD occlusion, followed by 4 weeks reperfusion. At the end of ischemia mice were injected with vehicle (Con) or IGF1 (40 ng/g ip), followed by continues treatment for 3 days via osmotic mini pumps (1 μg/g/d sc.). Cardiac function, determined by echocardiography at baseline and 1 and 4 weeks after ischemia, showed no differences at baseline in all mouse lines studied. IGF1 treatment improved cardiac function after 1 and 4 weeks in wild‐type mice ((EF, week 4: 49±4% (IGF1) vs 36±8% (Con)), but also in inducible cardiomyocyte‐specific IGF1‐receptor (IGF1R) KO mice (48±5% (IGF1) vs 35±5% (Con)). However, in myeloid cell specific IGF1R KO mice IGF1 was no longer protective (36±8% (IGF1) vs 38±8% (Con)), indicating that myeloid cells are responsible for the protective effect of IGF1 after MI. In addition, micro‐array analysis of the infarct area of the heart showed that mostly myeloid cell related pathways were altered in IGF treated mice 1 and 2 days after AMI. To look further into the mechanism involved, in vitro and subacute experiments were performed. FACS and qPCR analysis showed that treatment of monocyte derived macrophages with IGF1 induced macrophage polarization to macrophages with a reparative M2‐like phenotype, characterized by an increase in mannose receptor (CD206), arginase and resistin‐like α. Also, in vivo FACS analysis of the heart showed an increase in M2‐like (CD206 + ) macrophages in IGF1 treated mice 3 days after AMI (152 777±13510 (IGF1) vs 108 071±14023 (Con) cells per heart) without affecting total macrophage number. In these mice, no effects on the amount of lymphocytes or monocytes were observed in blood or heart. A trend towards an increased number of neutrophils in the heart was seen after IGF treatment (272 120±42478 (IGF1) vs 198 122±31741 (Con) cells per heart), without an effect on neutrophil amount in blood. In addition, one week after AMI, i.e. a timepoint where functional cardiac improvement was observed in echo analysis, histological analysis of scar size showed a significant reduction in scar size after IGF1 treatment (9.2±4% (IGF1) vs. 14.7±3.9% (Con) of left ventricle). This was accompanied by an increased vessel formation in the scar (654±132 (IGF1) vs 495±94 (Con) CD31 + cells/mm 2 ) and borderzone (1676±72 (IGF1) vs 1467±110 (Con)) after IGF1 treatment. Interestingly, short term no protective effects of IGF1 were observed. IGF1 treatment did not reduce infarct size 2 hours after AMI (38±10% (IGF1) vs. 39±11% (Con) of left ventricle). Short term IGF1 treatment improves cardiac function in the subacute phase after AMI by affecting myeloid cells. This effect was independent of the IGF1R in cardiomyocytes. IGF1 treatment increases the amount of reparative M2 macrophages, and may thereby improve vasculature and reduce scar size. Support or Funding Information Funded by SFB1116/A06 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Insulin-like growth factor 1 (IGF1) is an anabolic hormone that controls the growth and metabolism of many cell types. However, IGF1 also mediates cardio-protective effects after acute myocardial infarction (AMI), but the underlying mechanisms and cellular targets are not fully understood. Here we demonstrate that short-term IGF1 treatment for 3 days after AMI improved cardiac function after 1 and 4 weeks. Regional wall motion was improved in ischemic segments, scar size was reduced, and capillary density increased in the infarcted area and the border zone. Unexpectedly, inducible inactivation of the IGF1 receptor (IGF1R) in cardiomyocytes did not attenuate the protective effect of IGF1. Sequential cardiac transcriptomic analysis indicated an altered myeloid cell response in the acute phase after AMI, and, notably, myeloid-cell Igf1r-/- mice lost the protective IGF1 function after I/R. In addition, IGF1 induced an M2-like anti-inflammatory phenotype in bone marrow-derived macrophages and enhanced the number of anti-inflammatory macrophages in heart tissue on day 3 after AMI in vivo. In summary, modulation of the acute inflammatory phase after AMI by IGF1 represents an effective mechanism to preserve cardiac function after I/R.

Ciliopathies are life-threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans. Byperforming quantitative immunofluorescence studies in RPGRIP1L(-/-) mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type-specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate-specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.