Linagliptin prevents left ventricular stiffening by reducing titin cleavage and hypophosphorylation.

The metabolic syndrome (MetS) is an escalating problem worldwide, causing left ventricular stiffening, an early characteristic of diastolic dysfunction for which no treatment exists. As diastolic dysfunction and stiffening in MetS patients are associated with increased circulating dipeptidyl peptidase-4 (DPP-4) levels, we investigated whether the clinically approved DPP-4 inhibitor linagliptin reduces left ventricular stiffness in MetS-induced cardiac disease. Sixteen-week-old obese ZSF1 rats, displaying the MetS and left ventricular stiffness, received linagliptin-supplemented or placebo diet for four weeks. Linagliptin significantly reduced obesity, hyperlipidaemia, and hyperglycaemia and improved left ventricular relaxation. This improved relaxation was related to decreased cardiac fibrosis and cardiomyocyte passive stiffness (Fpassive ). The reduced Fpassive was the result of titin isoform switching from the stiff N2B to the more flexible N2BA and increased phosphorylation of total titin and specifically its N2Bus region (S4080 and S3391). Importantly, DPP-4 directly cleaved titin in vitro, resulting in an increased Fpassive , which was prevented by simultaneous administration of linagliptin. In conclusion, linagliptin improves left ventricular stiffness in obese ZSF1 rats by preventing direct DPP4-mediated titin cleavage, as well as by modulating both titin isoform levels and phosphorylation. Reducing left ventricular stiffness by administering linagliptin might prevent MetS-induced early diastolic dysfunction in human.

Microvascular and lymphatic dysfunction in HFpEF and its associated comorbidities.

Heart failure with preserved ejection fraction (HFpEF) is a complex heterogeneous disease for which our pathophysiological understanding is still limited and specific prevention and treatment strategies are lacking. HFpEF is characterised by diastolic dysfunction and cardiac remodelling (fibrosis, inflammation, and hypertrophy). Recently, microvascular dysfunction and chronic low-grade inflammation have been proposed to participate in HFpEF development. Furthermore, several recent studies demonstrated the occurrence of generalized lymphatic dysfunction in experimental models of risk factors for HFpEF, including obesity, hypercholesterolaemia, type 2 diabetes mellitus (T2DM), hypertension, and aging. Here, we review the evidence for a combined role of coronary (micro)vascular dysfunction and lymphatic vessel alterations in mediating key pathological steps in HFpEF, including reduced cardiac perfusion, chronic low-grade inflammation, and myocardial oedema, and their impact on cardiac metabolic alterations (oxygen and nutrient supply/demand imbalance), fibrosis, and cardiomyocyte stiffness. We focus primarily on HFpEF caused by metabolic risk factors, such as obesity, T2DM, hypertension, and aging.

Adipose tissue macrophages induce hepatic neutrophil recruitment and macrophage accumulation in mice.

OBJECTIVE: Obesity is a risk factor for non-alcoholic steatohepatitis (NASH). This risk has been attributed to visceral adipose tissue (vAT) expansion associated with increased proinflammatory mediators. Accumulation of CD11c+ proinflammatory adipose tissue macrophages (ATM) is an important driver of vAT inflammation. We investigated the role of ATMs in hepatic inflammation during NASH development. DESIGN: vAT isolated from lean, obese or ATM-depleted (using clodronate liposomes) obese mice was transplanted to lean ldlr-/- acceptor mice. Systemic and hepatic inflammation was assessed either after 2 weeks on standard chow or after 8 weeks on high cholesterol diet (HCD) to induce NASH. RESULTS: Transplanting donor vAT from obese mice increased HCD-induced hepatic macrophage content compared with lean-transplanted mice, worsening liver damage. ATM depletion prior to vAT transplantation reduced this increased hepatic macrophage accumulation. On chow, vAT transplantation induced a more pronounced increase in circulating and hepatic neutrophil numbers in obese-transplanted than lean-transplanted mice, while ATM depletion prior to vAT transplantation reversed this effect. Microarray analysis of fluorescence-activated cell sorting of CD11c+ and CD11c- macrophages isolated from donor adipose tissue showed that obesity resulted in enhanced expression of neutrophil chemotaxis genes specifically in CD11c+ ATMs. Involvement of the neutrophil chemotaxis proteins, CXCL14 and CXCL16, was confirmed by culturing vAT. In humans, CD11c expression in vAT of obese individuals correlated with vAT expression of neutrophil chemotactic genes and with hepatic expression of neutrophil and macrophage marker genes. CONCLUSION: ATMs from obese vAT induce hepatic macrophage accumulation during NASH development, possibly by enhancing neutrophil recruitment.

Sema3A promotes the resolution of cardiac inflammation after myocardial infarction.

Optimal healing after myocardial infarction requires not only the induction of inflammation, but also its timely resolution. In patients, 30 days post myocardial infarction, circulating monocytes have increased expression of Semaphorin3A (Sema3A) as compared to directly after admission. This increased expression coincides with increased expression of Cx3CR1-a marker of non-classical monocytes that are important for immune resolution hence proper wound healing. In mice, the expression of Sema3A also increases in response to myocardial ischemia being expressed by infiltrating leukocytes. Comparing Sema3A heterozygote (HZ) and wild type (WT) mice post myocardial infarction, revealed increased presence of leukocytes in the cardiac tissues of HZ mice as compared to WT, with no differences in capillary density, collagen deposition, cardiomyocyte surface area, chemokine-or adhesion molecules expression. Whilst infarct sizes were similar 14 days after myocardial infarction in both genotypes, Sema3A HZ mice had thinner infarcts and reduced cardiac function as compared to their WT littermates. In vitro experiments were conducted to study the role of Sema3A in inflammation and resolution of inflammation as a potential explanation for the differences in leukocyte recruitment and cardiac function observed in our in vivo experiments. Here, recombinant Sema3A protein was able to affect the pro-inflammatory state of cultured bone marrow derived macrophages. First, the pro-inflammatory state was altered by the induced apoptosis of classical macrophages in the presence of Sema3A. Second, Sema3A promoted the polarization of classical macrophages to resolution-phase macrophages and enhanced their efferocytotic ability, findings that were reflected in the infarcted cardiac tissue of the Sema3A HZ mice. Finally, we demonstrated that besides promoting resolution of inflammation, Sema3A was also able to retard the migration of monocytes to the myocardium. Collectively our data demonstrate that Sema3A reduces cardiac inflammation and improves cardiac function after myocardial infarction by promoting the resolution of inflammation.

Pericyte loss initiates microvascular dysfunction in the development of diastolic dysfunction.

Aims: Microvascular dysfunction has been proposed to drive heart failure with preserved ejection fraction (HFpEF), but the initiating molecular and cellular events are largely unknown. Our objective was to determine when microvascular alterations in HFpEF begin, how they contribute to disease progression, and how pericyte dysfunction plays a role herein. Methods and results: Microvascular dysfunction, characterized by inflammatory activation, loss of junctional barrier function, and altered pericyte-endothelial crosstalk, was assessed with respect to the development of cardiac dysfunction, in the Zucker fatty and spontaneously hypertensive (ZSF1) obese rat model of HFpEF at three time points: 6, 14, and 21 weeks of age. Pericyte loss was the earliest and strongest microvascular change, occurring before prominent echocardiographic signs of diastolic dysfunction were present. Pericytes were shown to be less proliferative and had a disrupted morphology at 14 weeks in the obese ZSF1 animals, who also exhibited an increased capillary luminal diameter and disrupted endothelial junctions. Microvascular dysfunction was also studied in a mouse model of chronic reduction in capillary pericyte coverage (PDGF-Bret/ret), which spontaneously developed many aspects of diastolic dysfunction. Pericytes exposed to oxidative stress in vitro showed downregulation of cell cycle-associated pathways and induced a pro-inflammatory state in endothelial cells upon co-culture. Conclusion: We propose pericytes are important for maintaining endothelial cell function, where loss of pericytes enhances the reactivity of endothelial cells to inflammatory signals and promotes microvascular dysfunction, thereby accelerating the development of HFpEF.

SARS-CoV-2 infection causes prolonged cardiomyocyte swelling and inhibition of HIF1α translocation in an animal model COVID-19.

Recovered COVID-19 patients often display cardiac dysfunction, even after a mild infection. Most current histological results come from patients that are hospitalized and therefore represent more severe outcomes than most COVID-19 patients face. To overcome this limitation, we investigated the cardiac effects of SARS-CoV-2 infection in a hamster model. SARS-CoV-2 infected hamsters developed diastolic dysfunction after recovering from COVID-19. Histologically, increased cardiomyocyte size was present at the peak of viral load and remained at all time points investigated. As this increase is too rapid for hypertrophic remodeling, we found instead that the heart was oedemic. Moreover, cardiomyocyte swelling is associated with the presence of ischemia. Fibrin-rich microthrombi and pericyte loss were observed at the peak of viral load, resulting in increased HIF1α in cardiomyocytes. Surprisingly, SARS-CoV-2 infection inhibited the translocation of HIF1α to the nucleus both in hamster hearts, in cultured cardiomyocytes, as well as in an epithelial cell line. We propose that the observed diastolic dysfunction is the consequence of cardiac oedema, downstream of microvascular cardiac ischemia. Additionally, our data suggest that inhibition of HIF1α translocation could contribute to an exaggerated response upon SARS-CoV-2 infection.

Towards standardization of echocardiography for the evaluation of left ventricular function in adult rodents: a position paper of the ESC Working Group on Myocardial Function.

Echocardiography is a reliable and reproducible method to assess non-invasively cardiac function in clinical and experimental research. Significant progress in the development of echocardiographic equipment and transducers has led to the successful translation of this methodology from humans to rodents, allowing for the scoring of disease severity and progression, testing of new drugs, and monitoring cardiac function in genetically modified or pharmacologically treated animals. However, as yet, there is no standardization in the procedure to acquire echocardiographic measurements in small animals. This position paper focuses on the appropriate acquisition and analysis of echocardiographic parameters in adult mice and rats, and provides reference values, representative images, and videos for the accurate and reproducible quantification of left ventricular function in healthy and pathological conditions.

Cellular and Molecular Differences between HFpEF and HFrEF: A Step Ahead in an Improved Pathological Understanding.

Heart failure (HF) is the most rapidly growing cardiovascular health burden worldwide. HF can be classified into three groups based on the percentage of the ejection fraction (EF): heart failure with reduced EF (HFrEF), heart failure with mid-range-also called mildly reduced EF- (HFmrEF), and heart failure with preserved ejection fraction (HFpEF). HFmrEF can progress into either HFrEF or HFpEF, but its phenotype is dominated by coronary artery disease, as in HFrEF. HFrEF and HFpEF present with differences in both the development and progression of the disease secondary to changes at the cellular and molecular level. While recent medical advances have resulted in efficient and specific treatments for HFrEF, these treatments lack efficacy for HFpEF management. These differential response rates, coupled to increasing rates of HF, highlight the significant need to understand the unique pathogenesis of HFrEF and HFpEF. In this review, we summarize the differences in pathological development of HFrEF and HFpEF, focussing on disease-specific aspects of inflammation and endothelial function, cardiomyocyte hypertrophy and death, alterations in the giant spring titin, and fibrosis. We highlight the areas of difference between the two diseases with the aim of guiding research efforts for novel therapeutics in HFrEF and HFpEF.

The effect of different anaesthetics on echocardiographic evaluation of diastolic dysfunction in a heart failure with preserved ejection fraction model.

Heart failure with preserved ejection fraction (HFpEF) is currently untreated. Therapeutics development demands effective diagnosis of diastolic dysfunction in animal models mimicking human pathology, which requires appropriate anaesthetics. Here, we investigated which anaesthetic, ketamine/xylazine or isoflurane, could be used to reveal diastolic dysfunction in HFpEF-diseased obese ZSF1 rats by echocardiography. First, diastolic dysfunction was confirmed by pressure-volume loops in obese compared to lean control ZSF1 rats. In echocardiography, ketamine/xylazine, unlike isoflurane, was able to demonstrate impaired relaxation in obese ZSF1 rats, as reflected by impaired early (E) and late (A) filling peak velocities, decreased E/A ratio, and a prolonged deceleration and isovolumic relaxation time. Interestingly, ketamine/xylazine induced a wider separation of both tissue and pulsed wave Doppler-derived echocardiographic waves required for diastolic dysfunction diagnosis, potentially by reducing the heart rate (HR), while isoflurane resulted in merged waves. To assess whether HR-lowering alone explained the differences between the anaesthetics, echocardiography measurements under isoflurane with and without the HR-lowering drug ivabradine were compared. However, diastolic dysfunction could not be diagnosed in ivabradine-treated obese ZSF1 rats. In summary, ketamine/xylazine compared to isoflurane is the anaesthetic of choice to detect diastolic dysfunction by echocardiography in rodent HFpEF, which was only partly mediated by HR-lowering.

Resistance to retinopathy development in obese, diabetic and hypertensive ZSF1 rats: an exciting model to identify protective genes.

Diabetic retinopathy (DR) is one of the major complications of diabetes, which eventually leads to blindness. Up to date, no animal model has yet shown all the co-morbidities often observed in DR patients. Here, we investigated whether obese 42 weeks old ZSF1 rat, which spontaneously develops diabetes, hypertension and obesity, would be a suitable model to study DR. Although arteriolar tortuosity increased in retinas from obese as compared to lean (hypertensive only) ZSF1 rats, vascular density pericyte coverage, microglia number, vascular morphology and retinal thickness were not affected by diabetes. These results show that, despite high glucose levels, obese ZSF1 rats did not develop DR. Such observations prompted us to investigate whether the expression of genes, possibly able to contain DR development, was affected. Accordingly, mRNA sequencing analysis showed that genes (i.e. Npy and crystallins), known to have a protective role, were upregulated in retinas from obese ZSF1 rats. Lack of retina damage, despite obesity, hypertension and diabetes, makes the 42 weeks of age ZSF1 rats a suitable animal model to identify genes with a protective function in DR. Further characterisation of the identified genes and downstream pathways could provide more therapeutic targets for the treat DR.