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Aims Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9−/− mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells. Methods and results Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9−/− mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9−/− hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9−/− mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9−/− mice. Circulating lipid levels were unchanged in CM-Pcsk9−/− mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9−/− mice had an increased number of mitochondria–endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism. Conclusion PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production.

Aims Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function. Methods and results Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg–/– mice). In 9- to 10-week-old hUgcg–/– mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg–/– mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to β-adrenergic stimulation was reduced in cardiomyocytes from hUgcg–/– mice and that Ugcg knockdown suppressed the internalization and trafficking of β1-adrenergic receptors. Conclusions Our findings suggest that cardiac glycosphingolipids are required to maintain β-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function., Graphical Abstract Cardiac glycosphingolipids are required to maintain β-adrenergic signaling and contractile capacity in cardiomyocytes and to preserve normal heart function.

The adult heart develops hypertrophy to reduce ventricular wall stress and maintain cardiac function in response to an increased workload. Although pathological hypertrophy generally progresses to heart failure, physiological hypertrophy may be cardioprotective. Cardiac-specific overexpression of the lipid-droplet protein perilipin 5 (Plin5) promotes cardiac hypertrophy, but it is unclear whether this response is beneficial. We analyzed RNA-sequencing data from human left ventricle and showed that cardiacPLIN5expression correlates with up-regulation of cardiac contraction–related processes. To investigate how elevated cardiac Plin5 levels affect cardiac contractility, we generated mice with cardiac-specific overexpression ofPlin5(MHC-Plin5mice). These mice displayed increased left ventricular mass and cardiomyocyte size but preserved heart function. Quantitative proteomics identified sarcoplasmic/endoplasmic reticulum Ca2+ATPase 2 (SERCA2) as a Plin5-interacting protein. In situ proximity ligation assay further confirmed the Plin5/SERCA2 interaction. Live imaging showed increases in intracellular Ca2+release during contraction, Ca2+removal during relaxation, and SERCA2 function in MHC-Plin5versus WT cardiomyocytes. These results identify a role of Plin5 in improving cardiac contractility through enhanced Ca2+signaling.

Endothelial injury makes the vessel wall vulnerable to cardiovascular diseases. Injured endothelium regenerates by collective sheet migration, that is, the endothelial cells coordinate their motion and regrow as a sheet of cells with retained cell-cell contacts into the wounded area. Leukocytes appear to be involved in endothelial repair in vivo; however, little is known about their identity and role in the reparative sheet migration process. To address these questions, we developed a high-quality en face technique that enables visualizing of leukocytes and endothelial cells simultaneously following an endoluminal scratch wound injury of the mouse carotid artery. We discovered that regrowing endothelium forms a broad proliferative front accompanied by CD11c+ leukocytes. Functionally, the leukocytes were dispensable for the initial migratory response of the regrowing endothelial sheet, but critical for the subsequent formation and maintenance of a front zone with high cellular density. Marker expression analyses, genetic fate mapping, phagocyte targeting experiments, and mouse knock-out experiments indicate that the CD11c+ leukocytes were mononuclear phagocytes with an origin from both Ly6Chigh and Ly6Clow monocytes. In conclusion, CD11c+ mononuclear phagocytes are essential for a proper endothelial regrowth following arterial endoluminal scratch injury. Promoting the endothelial-preserving function of CD11c+ leukocytes may be a strategy to enhance endothelial repair following surgical and endovascular procedures. CD11c+ mononuclear phagocytes are recruited specifically to regenerating endothelium following vascular injury to orchestrate endothelial sheet migration.

The microsomal triglyceride transport protein (MTTP) is critical for assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins and is most abundant in the liver and intestine. Surprisingly, MTTP is also expressed in the heart. Here we tested the functional relevance of cardiac MTTP expression.We combined clinical studies, advanced expression analysis of human heart biopsies and analyses in genetically modified mice lacking cardiac expression of the MTTP-A isoform of MTTP.Our results indicate that lower cardiac MTTP expression in humans is associated with structural and perfusion abnormalities in patients with ischemic heart disease. MTTP-A deficiency in mice heart does not affect total MTTP expression, activity or lipid concentration in the heart. Despite this, MTTP-A deficient mice displayed impaired cardiac function after a myocardial infarction. Expression analysis of MTTP indicates that MTTP expression is linked to cardiac function and responses in the heart.Our results indicate that MTTP may play an important role for the heart function in conjunction to ischemic events.

Endothelial injury makes the vessel wall vulnerable to cardiovascular diseases. Injured endothelium regenerates by collective sheet migration, that is, the endothelial cells coordinate their motion and regrow as a sheet of cells with retained cell-cell contacts into the wounded area. Leukocytes appear to be involved in endothelial repair in vivo; however, little is known about their identity and role in the reparative sheet migration process. To address these questions, we developed a high-quality en face technique that enables visualizing of leukocytes and endothelial cells simultaneously following an endoluminal scratch wound injury of the mouse carotid artery. We discovered that regrowing endothelium forms a broad proliferative front accompanied by CD11c

IGF-1 plays an important role in cardiovascular homeostasis, and plasma levels of IGF-1 correlate inversely with systolic function in heart failure. It is not known to what extent circulating IGF-1 secreted by the liver and local autocrine/paracrine IGF-1 expressed in the myocardium contribute to these beneficial effects on cardiac function and morphology. In the present study, we used a mouse model of liver-specific inducible deletion of the IGF-1 gene (LI-IGF-1 -/- mouse) in an attempt to evaluate the importance of circulating IGF-I on cardiac morphology and function under normal and pathological conditions, with an emphasis on its regulatory role in myocardial phosphocreatine metabolism. Echocardiography was performed in LI-IGF-1 -/- and control mice at rest and during dobutamine stress, both at baseline and post myocardial infarction (MI). High-energy phosphate metabolites were compared between LI-IGF-1 -/- and control mice at 4 weeks post MI. We found that LI-IGF-1 -/- mice had significantly greater left ventricular dimensions at baseline and showed a greater relative increase in cardiac dimensions, as well as deterioration of cardiac function, post MI. Myocardial creatine content was 17.9% lower in LI-IGF-1 -/- mice, whereas there was no detectable difference in high-energy nucleotides. These findings indicate an important role of circulating IGF-1 in preserving cardiac structure and function both in physiological settings and post MI.

The heart produces apolipoprotein (apo) B-containing lipoproteins which enables cardiac export of potentially cardiotoxic lipids. We hypothesized that overexpression of apoB attenuates the pathologic cardiac remodeling and hypertrophic response following pathological stimuli such as chronic adrenergic overstimulation and myocardial infarction (MI).Cardiac hypertrophy was induced by a chronic infusion of isoproterenol (ISO) 15 mg/kg/day for 3 weeks in human apoB transgenic mice (n = 9) and in non-transgenic wild-type mice (n = 10). As controls, apoB transgenic (n = 10) and wild-type mice (n = 10) saline infusions were used. Transthoracic echocardiography was performed at baseline and after 3 weeks of treatment to evaluate left ventricular (LV) function and morphology. To investigate the effects of expression on postinfarct hypertrophic response we induced MI in apoB transgenic mice (n = 8) and in wild-type controls (n = 11). The hearts were explanted and weighed 6 weeks post MI.At baseline, WT mice had higher BW and LV mass (LVM) compared to the apoB mice. The increase in LV mass and dimensions after 3 weeks of treatment with ISO was significantly lower while systolic function was significantly better in the apoB group. Six weeks post MI the apoB mice had significantly lower heart weight and heart weight to body weight ratio. The infarct size was similar in both groups.Overexpression of apoB attenuates the pathologic remodeling and hypertrophic response to chronic adrenergic stimulation and MI. Our results indicate that cardiac expression of apoB-containing lipoproteins might be an important regulator of myocardial structure and function.

Doxorubicin (DOX) is an effective antitumour agent against a variety of human malignancies but is associated with deleterious side effects, including myocardial damage and heart failure. Myocardial apoB-containing lipoprotein (apoB) is upregulated post myocardial infarction and has been shown to be cardioprotective in this setting by unloading excessive lipid. The aim of this study was to investigate whether apoB expression is increased also in DOX-induced heart failure and whether apoB overexpression protects the heart in DOX-induced myocardial injury.Cardiac function and energy metabolism was studied in mice and rats 24 hours after intraperitoneally administered DOX.We found that the content of apoB was decreased in rat myocardium 24 hours after DOX injection. In contrast, apoB content was increased in the infarcted myocardium of rats 24 hours post ischemia-reperfusion. Moreover, transgenic mice overexpressing apoB had better cardiac function and lower intracellular lipid accumulation compared to wild type mice 24 hours post DOX.Our findings indicate that depression of the myocardial apoB system may contribute to DOX-induced cardiac injury and that overexpression of apoB is protective, not only in ischemically damaged myocardium, but also in DOX-induced heart failure.

The failing heart is characterized by disturbed myocardial energy metabolism and creatine depletion. The aims of this study were to evaluate in vivo the effects of creatine (Cr) depletion on 1) left ventricular (LV) function, morphology, and lipid metabolism and 2) to test whether functional, morphologic, and metabolic disturbances induced by Cr depletion are reversible.Male Balb/c mice approximately 20 g were used. Two groups were studied: the mice treated with creatine analogue beta-guanidinopropionic acid (BGP) (n = 30) and controls (n = 30). BGP (1 M) were administered by subcutaneously implanted osmotic minipumps for 4 weeks. The mice were examined in vivo using echocardiography. High-performance liquid chromatography was used for measurements of the myocardial creatine, adenosine nucleotides, and lipids. BGP was discontinued in a subgroup of mice and these animals were followed for an additional 4 weeks, after which echocardiography was performed under resting and stress conditions. Body weight was lower in BGP mice (P.001) compared with the controls after 4 weeks. The total myocardial Cr pool was approximately 40% lower (P.001), whereas total nucleotide pool (TAN) was 18% lower (P = n.s.) in the BGP group. LV systolic function was disturbed at rest and stress in the BGP mice (both P.05). LV dimensions and LV mass were increased in the BGP group (P.05). There was an accumulation of intracellular triglycerides in the BGP-treated mice (P.05). Four weeks after BGP discontinuation Cr, TAN and TG content were restored to the normal levels while LV function, dimension, and mass were normalized.Myocardial Cr depletion results in LV dysfunction, pathologic remodeling, and lipid accumulation. These alterations are completely reversible on normalization of Cr content. Cr metabolism may be an important target for pharmacologic intervention to increase myocardial efficiency and structural integrity of the failing heart.

Aims In myocardial ischaemia, vascular endothelial growth factor (VEGF) induces permeability by activating a signalling pathway that includes VEGF receptor 2 (VEGFR2), resulting in increased oedema and inflammation and thereby expanding the area of tissue damage. In this study, we investigated the role of receptor-interacting protein 2 (Rip2) in VEGF signalling and myocardial ischaemia/reperfusion injury. Methods and results To determine whether Rip2 has a role in VEGF signalling, we used cultured endothelial cells in which Rip2 was or was not inactivated. In Rip2-deficient endothelial cells, stimulation with VEGF resulted in more rapid kinetics of VEGFR2 phosphorylation than in control cells. Rip2 deficiency also enhanced VEGF-induced activation of ERK1/2, suggesting an increased propensity for endothelial permeability. In a mouse model of myocardial ischaemia, Rip2 deficiency resulted in enhanced vascular permeability, increased oedema and expanding area of myocardial damage, and markedly reduced heart function after long-term follow-up. Conclusion Our results show that Rip2 modifies VEGF-induced signalling and vascular permeability in myocardial ischaemia. These findings indicate that Rip2 may be a promising novel therapeutic target to reduce excess vascular permeability in ischaemic heart disease.

Impaired cardiac function is associated with myocardial triglyceride accumulation, but it is not clear how the lipids accumulate or whether this accumulation is detrimental. Here we show that hypoxia/ischemia-induced accumulation of lipids in HL-1 cardiomyocytes and mouse hearts is dependent on expression of the VLDL receptor (VLDLR). Hypoxia-induced VLDLR expression in HL-1 cells was dependent on HIF-1α through its interaction with a hypoxia-responsive element in the Vldlr promoter, and VLDLR promoted the endocytosis of lipoproteins. Furthermore, VLDLR expression was higher in ischemic compared with nonischemic left ventricles from human hearts and was correlated with the total lipid droplet area in the cardiomyocytes. Importantly, Vldlr-/- mice showed improved survival and decreased infarct area following an induced myocardial infarction. ER stress, which leads to apoptosis, is known to be involved in ischemic heart disease. We found that ischemia-induced ER stress and apoptosis in mouse hearts were reduced in Vldlr-/- mice and in mice treated with antibodies specific for VLDLR. These findings suggest that VLDLR-induced lipid accumulation in the ischemic heart worsens survival by increasing ER stress and apoptosis.

The heart produces apolipoprotein-B containing lipoproteins (apoB) whose function is not well understood. The aim of this study was to evaluate importance of myocardial apoB for cardiac function, structure and survival in myocardial infarction (MI) and heart failure (HF).MI was induced in mice (n=137) and myocardial apoB content was measured at 30 min, 3, 6, 24, 48, 120 h and 8 weeks post-MI. Transgenic mice overexpressing apoB (n=27) and genetically matched controls (n=27) were used to study the effects of myocardial apoB on cardiac function, remodeling, arrhythmias and survival after MI. Echocardiography was performed at rest and stress conditions at baseline, 2, 4 and 6 week post-MI and cumulative survival rate was registered. The myocardial apoB content increased both in the injured and the remote myocardium (p0.05) in response to ischemic injury. ApoB mice had 2-fold higher survival rate (p0.05) and better systolic function (p0.05) post-MI.Overexpression of apoB in the heart increases survival and improves cardiac function after acute MI. Myocardial apoB may be an important cardioprotective system in settings such as myocardial ischemia and HF.

Introduction: Human embryonic stem cells (hES) have emerged as an attractive and promising new therapeutic approach for treatment of heart diseases. The aim of this study was to evaluate in rats with acute myocardial infarction (MI): 1) effects of myocardial transplantation of undifferentiated hES on left ventricular (LV) function and morphology and 2) to determine whether potential improvement in LV function requires the presence of hES in the myocardium. Methods:Male Sprague-Dawley rats∼200 g were used. MI was induced by cryo-injury (protocol 1) and by ligation of left coronary artery (protocol 2). These procedures resulted in anterior MI engaging ∼30-40% of left ventricle (LV). In the protocol 1, the rats were randomized into two groups: rats with MI treated with vehicle (n = 8) and rats with MI treated with 1 million hES cells (n = 8). Rats treated with hES cells received cyclosporine (5 mg/kg/d). The hES cells were suspended in 0,05 ml buffer and transplanted by intramyocardial injection into the viable myocardium close to the infracted area directly after cryo-injury. The rats were followed for 1 week. In order to evaluate hypothetical paracrine effects of hES, in the protocol 2, the rats were randomised into three groups: rats with MI treated with vehicle (n = 4), rats with MI treated with cell medium (n = 6) and rats with MI treated with supernatant extracted from hES cell culture (n = 6). The animals received i.p. injections 3 times/week during 4 weeks. All animals were investigated with transthoracal echocardiography, continuous ECG and LV catheterization. Post-mortem, the hearts were evaluated histologically. Results: In the protocol 1 neither deaths nor arrhythmias occurred in the rats treated with hES cells. dP/dT was similar in both groups. There were no signs of abnormal tissue growth at the site of hES cell engraftment after one week. There was no difference in the indices of LV systolic function while diastolic function was significantly improved in the hES rats (p < 0.05). hES were detected in 1/8 rats in the infarcted area. In the protocol 2, no difference was found between the groups in indices of LV function and morphology after 4 weeks of treatment. Conclusion: Transplantation of undifferentiated hES cells have positive effect on LV diastolic function in the rat model of acute MI. This effect requires the presence of hES in the tissue. The use of hES may be an important approach for cardio-reparation and reconstitution of normal cardiac structure and function in the future.

Cardiac reserve can be used to predict survival and outcome in patients with heart failure. The aim of this study was to investigate if native cardiac reserve could predict survival after myocardial infarction (MI) in mice. Method We investigated 27 healthy C57Bl6 mice (♂10–12 weeks old) with echocardiography using a high-frequency 15-MHz linear transducer. Investigations were performed both at rest and after pharmacological stress induced by dobutamine (1 μg/g body weight i.p.). The day after the echocardiography examination, a large MI was induced by ligation of the left anterior descending (LAD) coronary artery for evaluation of mortality rate. Results Two weeks after induction of MI, 7 mice were alive (26%). Evaluation of the difference between the surviving and deceased animals showed that the survivors had a better native ability to increase systolic performance (ΔLVESd -1.86 vs -1.28mm p = 0.02) upon dobutamine challenge, resulting in a better cardiac reserve (ΔFS 37 vs 25% p = 0.02 and ΔCO 0.27 vs -0.10 ml/min p = 0.02) and a better chronotropic reserve (ΔR-R interval -68 vs -19 ms p < 0.01). A positive relationship was found between ability to survive and both cardiac (p < 0.05) and chronotropic reserve (p < 0.05) when the mice were divided into three groups: survivors, surviving < 7 days, and surviving < 1 day. Conclusion We conclude that before MI induction the surviving animals had a better cardiac function compared with the deceased. This indicates that native cardiac and chronotropic reserve may be an important determinant and predictor of survival in the setting of large MI and post-infarction heart failure.