AIMS:Myocardial infarction (MI) remains one of the leading causes of mortality and morbidity worldwide. Cardiac remodelling is a key process following MI, involving changes in cellular composition and extracellular matrix (ECM) to adapt to injury. However, maladaptive remodelling can worsen cardiac function, leading to cardiac fibrosis and heart failure. In the context of MI, cell migration inducing protein (CEMIP) has come into focus and its ability to modulate hyaluronan (HA) turnover has raised critical questions about its role in post-MI healing. METHODS AND RESULTS:This study investigates the role of global CEMIP deletion in a murine closed chest ischaemia and reperfusion injury (I/R) model. We demonstrate that Cemip is significantly upregulated in the infarcted area of the heart peaking at 72 h post-I/R. Furthermore, global deletion of Cemip resulted in significantly impaired cardiac function post-I/R accompanied by an increased scar size, cardiac collagen content, and PERIOSTIN deposition. Flow cytometric analyses revealed increased cardiac fibroblast abundance driven by a decrease in apoptosis at 72 h post-I/R. In contrast, cardiac fibroblast proliferation was strongly inhibited in Cemip-KO fibroblasts. Upregulation of myofibroblast-associated genes as well as morphological changes in Cemip-KO cardiac fibroblasts pointed towards a crucial role of CEMIP in controlling fibroblast to myofibroblast conversion. Single-cell RNA sequencing of infarcted hearts confirmed upregulation of pro-fibrotic genes in Cemip-KO myofibroblasts. Mechanistically, Cemip-deficiency appears to maintain TGF-β activity in cardiac fibroblasts by enhancing integrin-mediated latent TGF-β activation and constitutive SMAD3 activation. This might foster a pro-fibrotic gene expression programme leading to improved fibroblast survival, aberrant activation and myofibroblast persistence post-I/R. CONCLUSION:Our data suggest that CEMIP contributes to post-infarct healing by limiting excessive activation and fibroblast to myofibroblast differentiation during scar formation. Thus, CEMIP may be considered as a novel target to prevent maladaptive cardiac fibrosis and heart failure.

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.

Heart disease is characterized by stress-induced endoreplication preceding pathological cardiomyocyte overgrowth, yet the upstream regulatory mechanisms linking tissue hypoxia to aberrant cellular growth remain incompletely defined. Here, we identify cardiac hypoxia as a key determinant of endoreplication through activation of a hypoxia-inducible factor-1 alpha-microRNA regulatory axis that converges on mitochondrial energetic control. We show that stress-induced activation of hypoxia-inducible factor-1 alpha drives transcriptional induction of microRNA-27b-5p, which directly represses the ATP synthase subunit ATP5A1, resulting in impaired mitochondrial ATP synthesis and accumulation of intra-mitochondrial ADP. Elevated ADP serves as a rate-limiting cofactor for one-carbon metabolism, promoting formate production and de novo purine biosynthesis, thereby enabling pathological endoreplication and cardiomyocyte hypertrophic growth. Genetic gain- and loss-of-function studies targeting hypoxia-inducible factor-1 alpha, microRNA-27b, and ATP5A1 across multiple mouse models of cardiac stress, together with correlative analyses of human cardiac biopsies, establish a conserved and causal relationship between dysregulated mitochondrial energetics and pathological cardiac remodeling. Inhibition of microRNA-27b-5p attenuates established cardiac hypertrophy, improves cardiac function, and suppresses stress-induced multinucleation in vivo. Leveraging this mechanistic insight, we identify the clinically approved antifolate compound methotrexate as an effective inhibitor of stress-induced cardiac endoreplication and pathological hypertrophy in preclinical models. Collectively, these findings define a druggable hypoxia-driven metabolic pathway linking mitochondrial ATP homeostasis to pathological cardiomyocyte growth and suggest therapeutic opportunities for targeting maladaptive cardiac remodeling.

Adipose tissue ATGL has emerged as an important player in cardiovascular disease. Myocardial infarction is accompanied by sympathetic stimulation and activation of white adipose tissue and peripheral lipolysis. We therefore investigate here the role of adipocyte ATGL in a murine model of cardiac ischemia and reperfusion (I/R) by using an inducible, adipocyte specific KO of ATGL (iatATGL-KO). Notably this led to successfully inhibited lipolysis during cardiac ischemia, and KO mice exhibited aggravated cardiac dysfunction and enhanced scar formation after 28 days I/R. This phenotype went along with multiple structural and molecular alterations mainly in the subcutaneous white adipose tissue depot (iWAT) and brown adipose tissue (BAT). The iatATGL-KO mainly reduced BAT activation as well as adiponectin-secretion. In the heart spatial transcriptomic analysis suggested higher mechanical stress in the remote myocardium, which went along with higher oxygen consumption rates (OCR) and higher dependency on glucose as substrate after 24 h I/R. Taken together, iatATGL-KO hearts after I/R seem to be affected in multiple ways, such as a reduction in cardioprotective factors from iWAT and BAT as well as an oxygen wasting effect in the remote zone of the heart, which contribute to the worse outcome. This indicates a time and depot-specific role of adipocyte ATGL in cardiac ischemia and reperfusion injury.

Despite its promise, cardiac regenerative therapy remains clinically elusive due to the difficulty of spatio-temporal control of proliferative induction, and the need to coordinately reprogram multiple regulatory pathways to overcome the strict post-mitotic state of human adult cardiomyocytes. To address this unmet therapeutic need, a combinatorial miRNA interference screen is performed specifically targeting cardiac-predominant miRNAs regulating key aspects of cardiomyocyte mitotic induction to cell-cycle completion in neonatal rat cardiomyocytes. In doing so combinatorial interference of miRNA-1a and miRNA-15b (LNA-1a/15b) is identified as drivers of adult cardiomyocyte proliferation. Due to miRNA-1a/15b function on multiple processes modulating adult cardiomyocyte mitosis, its inhibition augmented adult cardiomyocyte cell-cycle completion and daughter cell formation, and improved contractility in 3D human cardiac organoids, and in a mouse model of ST-segment elevation myocardial infarction. Due to the cardiac-restricted pattern of miRNA-1a/15b expression, this strategy provides a feasible means for specific cardiomyocyte proliferative induction with minimal risk of neoplasm formation and off-target toxicity. The approach further highlights an underutilized therapeutic strategy for simultaneous co-regulation of multiple disease pathways through combinatorial interference of miRNAs.

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.

Obesity is one of the diseases with severe health consequences and rapidly increasing worldwide prevalence. Understanding the complex network of food intake and energy balance regulation is an essential prerequisite for pharmacological intervention with obesity. G protein-coupled receptors (GPCRs) are among the main modulators of metabolism and energy balance. They, for instance, regulate appetite and satiety in certain hypothalamic neurons, as well as glucose and lipid metabolism and hormone secretion from adipocytes. Mutations in some GPCRs, such as the melanocortin receptor type 4 (MC4R), have been associated with early-onset obesity. Here, we identified the adhesion GPCR latrophilin 1 (ADGRL1/LPHN1) as a member of the regulating network governing food intake and the maintenance of energy balance. Deficiency of the highly conserved receptor in mice results in increased food consumption and severe obesity, accompanied by dysregulation of glucose homeostasis. Consistently, we identified a partially inactivating mutation in human ADGRL1/LPHN1 in a patient suffering from obesity. Therefore, we propose that LPHN1 dysfunction is a risk factor for obesity development.

BACKGROUND Despite its promise, cardiac regenerative therapy remains clinically elusive due to the difficulty of spatio-temporal control of proliferative induction, and the need to coordinately reprogram multiple regulatory pathways to overcome the strict post-mitotic state of human adult cardiomyocytes. The present study was designed to identify a novel combinatorial miRNA therapy to address this unmet therapeutic need.METHODS We performed a combinatorial miRNA interference screen specifically targeting cardiac-predominant miRNAs regulating key aspects of cardiomyocyte mitotic induction to cell-cycle completion, including sarcomerogenesis, metabolic and cell-cycle control pathways. Cardiomyocyte proliferation and cardiac function were assessed in human cardiac biopsies, human cardiac tissue mimetics and in mouse disease models.RESULTS We identified combinatorial interference of miR-1a and miR-15b (LNA-1a/15b) as drivers of adult cardiomyocyte proliferation. Due to miR-1a/15b function on multiple processes modulating adult cardiomyocyte mitosis, its inhibition augmented adult cardiomyocyte cell-cycle completion and daughter cell formation, and improved contractility in in vitro 2D and 3D ischemic models, and in a mouse model of ST-segment elevation myocardial infarction (STEMI). Due to the cardiac-restricted pattern of miR-1a/15b expression, this strategy provides a feasible strategy for specific cardiomyocyte proliferative induction with minimal risk of neoplasm formation and off-target toxicity.CONCLUSIONS Combinatorial miR-1a/15b inhibition drives mitotic re-entry in adult cardiomyocytes and improves cardiac function in response to myocardial infarction. Our data provides a novel and clinically feasible LNA-based anti-miR-1a/15b strategy to attenuate heart failure and highlights an underutilized therapeutic strategy for simultaneous co-regulation of multiple disease pathways through combinatorial miRNA interference.### Competing Interest StatementT.Y., S.D. and J.K. are inventors on a patent application pertaining to the inhibition of miR-1a and miR-15b for the treatment of heart disease.### Funding StatementThis work was supported by the Messer Foundation, LOEWE Center for Cell and Gene Therapy and the European Innovation Council (GA: 822455) to J.K., and the SFB-TRR 267 (Non-coding RNA in the cardiovascular system) and the German Research Foundation (DFG) (Exc2026) to S.D. and J.K., and instrument grant support (INST 515/28-1 FUGG) to M.P., the European Research Council (Angiolnc) to S.D., and the Foundation for Pathobiochemistry and Molecular Diagnostics to P.M. Y.W. was supported by the China Scholarship Council (CSC) Grant #202108080020.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ethics committee of Goethe University Frankfurt Research Services gave ethical approval for this work. All necessary patient/participant consent has been obtained and patient/participant/sample identifiers cannot be used to identify individuals. Ethics committee of Regierungsprasidium Darmstadt gave ethical approval for the animal studies this work.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors.

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.

Although p38 MAP Kinase α (p38 MAPKα) is generally accepted to play a central role in the cardiac stress response, to date its function in maladaptive cardiac hypertrophy is still not unambiguously defined. To induce a pathological type of cardiac hypertrophy we infused angiotensin II (AngII) for 2 days via osmotic mini pumps in control and tamoxifen-inducible, cardiomyocyte (CM)-specific p38 MAPKα KO mice (iCMp38αKO) and assessed cardiac function by echocardiography, complemented by transcriptomic, histological, and immune cell analysis. AngII treatment after inactivation of p38 MAPKα in CM results in left ventricular (LV) dilatation within 48 h (EDV: BL: 83.8 ± 22.5 µl, 48 h AngII: 109.7 ± 14.6 µl) and an ectopic lipid deposition in cardiomyocytes, reflecting a metabolic dysfunction in pressure overload (PO). This was accompanied by a concerted downregulation of transcripts for oxidative phosphorylation, TCA cycle, and fatty acid metabolism. Cardiac inflammation involving neutrophils, macrophages, B- and T-cells was significantly enhanced. Inhibition of adipose tissue lipolysis by the small molecule inhibitor of adipocytetriglyceride lipase (ATGL) Atglistatin reduced cardiac lipid accumulation by 70% and neutrophil infiltration by 30% and went along with an improved cardiac function. Direct targeting of neutrophils by means of anti Ly6G-antibody administration in vivo led to a reduced LV dilation in iCMp38αKO mice and an improved systolic function (EF: 39.27 ± 14%). Thus, adipose tissue lipolysis and CM lipid accumulation augmented cardiac inflammation in iCMp38αKO mice. Neutrophils, in particular, triggered the rapid left ventricular dilatation. We provide the first evidence that p38 MAPKα acts as an essential switch in cardiac adaptation to PO by mitigating metabolic dysfunction and inflammation. Moreover, we identified a heart–adipose tissue–immune cell crosstalk, which might serve as new therapeutic target in cardiac pathologies.

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.

Diabetes mellitus type 2 is associated with adverse clinical outcome after myocardial infarction. To better understand the underlying causes we here investigated sarcomere protein function and its calcium-dependent regulation in the non-ischemic remote myocardium (RM) of diabetic mice (db/db) after transient occlusion of the left anterior descending coronary artery. Before and 24 h after surgery db/db and non-diabetic db/+ underwent magnetic resonance imaging followed by histological and biochemical analyses of heart tissue. Intracellular calcium transients and sarcomere function were measured in isolated cardiomyocytes. Active and passive force generation was assessed in skinned fibers and papillary muscle preparations. Before ischemia and reperfusion (I/R), beat-to-beat calcium cycling was depressed in diabetic cardiomyocytes. Nevertheless, contractile function was preserved owing to increased myofilament calcium sensitivity and higher responsiveness of myocardial force production to β-adrenergic stimulation in db/db compared to db/+. In addition, protein kinase C activity was elevated in db/db hearts leading to strong phosphorylation of the titin PEVK region and increased titin-based tension of myofilaments. I/R impaired the function of whole hearts and RM sarcomeres in db/db to a larger extent than in non-diabetic db/+, and we identified several reasons. First, the amplitude and the kinetics of cardiomyocyte calcium transients were further reduced in the RM of db/db. Underlying causes involved altered expression of calcium regulatory proteins. Diabetes and I/R additively reduced phospholamban S16-phosphorylation by 80% (P < 000.1) leading to strong inhibition of the calcium ATPase SERCA2a. Second, titin stiffening was only observed in the RM of db/+, but not in the RM of db/db. Finally, db/db myofilament calcium sensitivity and force generation upon β-adrenergic stimulation were no longer enhanced over db/+ in the RM. The findings demonstrate that impaired cardiomyocyte calcium cycling of db/db hearts is compensated by increased myofilament calcium sensitivity and increased titin-based stiffness prior to I/R. In contrast, sarcomere function of the RM 24 h after I/R is poor because both these compensatory mechanisms fail and myocyte calcium handling is further depressed.

The incidence of heart failure after myocardial infarction (MI) remains high and the underlying causes are incompletely understood. The crosstalk between heart and adipose tissue and stimulated lipolysis has been identified as potential driver of heart failure. Lipolysis is also activated acutely in response to MI. However, the role in the post-ischemic remodeling process and the contribution of different depots of adipose tissue is unclear. Here, we employ a mouse model of 60 min cardiac ischemia and reperfusion (I/R) to monitor morphology, cellular infiltrates and gene expression of visceral and subcutaneous white adipose tissue depots (VAT and SAT) for up to 28 days post ischemia. We found that in SAT but not VAT, adipocyte size gradually decreased over the course of reperfusion and that these changes were associated with upregulation of UCP1 protein, indicating white adipocyte conversion to the so-called ‘brown-in-white’ phenotype. While this phenomenon is generally associated with beneficial metabolic consequences, its role in the context of MI is unknown. We further measured decreased lipogenesis in SAT together with enhanced infiltration of MAC-2+ macrophages. Finally, quantitative PCR analysis revealed transient downregulation of the adipokines adiponectin, leptin and resistin in SAT. While adiponectin and leptin have been shown to be cardioprotective, the role of resistin after MI needs further investigation. Importantly, all significant changes were identified in SAT, while VAT was largely unaffected by MI. We conclude that targeted interference with lipolysis in SAT may be a promising approach to promote cardiac healing after ischemia.

Cardiac metabolic remodeling is one of the early changes driving the progression of heart failure and is present even before overt cardiac dysfunction. Previously we showed that the tamoxifen inducible deletion of cardiomyocyte specific p38 MAPKa (KO) leads to a pronounced left ventricular dilation with a strongly impaired heart function just within 2 days of angiotensin II (ANGII) treatment. Additionally, a strong infiltration of immune cells in the cardiac tissue can be observed.