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Aims The proportion of patients hospitalized for heart failure (HF) with preserved left ventricular ejection fraction (LVEF) is rising, but no approved treatment exists, in part owing to incomplete characterization of this particular HF phenotype. In order to better define the characteristics of HF phenotypes in Finland, a large cohort with 12 years' follow‐up time was analysed. Methods and results Patients diagnosed between 2005 and 2017 at the Hospital District of Southwest Finland were stratified according to LVEF measure and N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) levels. For this retrospective registry study, previously diagnosed HF patients were defined as follows: patients with reduced ejection fraction (HFrEF; LVEF ≤ 40%; n = 4042), mid‐range ejection fraction (HFmrEF; LVEF > 40–50% and NT‐proBNP ≥ 125 pg/mL; n = 1468), and preserved ejection fraction (HFpEF; LVEF > 50% and NT‐proBNP ≥ 125 pg/mL; n = 3122) and followed up for 15 022, 4962, and 10 097 patient‐years, respectively. Cardiovascular (CV) hospitalization and mortality, influence of pre‐selected covariates on hospitalization and mortality, and the proportion of HFpEF and HFmrEF patients with a drop in LVEF to HFrEF phenotype were analysed. All data were extracted from the electronic patient register. HFrEF patients were rehospitalized slightly earlier than HFpEF/HFmrEF patients, but the second, third, and fourth rehospitalization rates did not differ between the subgroups. Female gender and better kidney function were associated with reduced rehospitalizations in HFmrEF and HFrEF, with a non‐significant trend in HFpEF. Each additional hospitalization was associated with a two‐fold increased risk of death and 2.2‐ to 2.3‐fold increased risk of CV death. All‐cause mortality was higher in patients with HFpEF. Although CV mortality was less frequent in HFpEF patients, it was associated with increased NT‐proBNP concentrations at index in all patient groups. During the 10 years following the index date, 26% of HFmrEF patients and 10% of HFpEF patients progressed to an HFrEF phenotype. Conclusions These findings suggest that disease progression, in terms of increased frequency of hospitalizations, and the relationship between increased number of hospitalizations and mortality are similar by LVEF phenotypes. These data highlight the importance of effective treatments that can reduce hospitalizations and suggest a role for monitoring NT‐proBNP levels in the management of HFpEF patients in particular.

Background and aims Dipeptidyl peptidase 4 (DPP-4) inhibitors have anti-inflammatory and atheroprotective effects. We evaluated the effects of the DPP-4 inhibitor linagliptin on atherosclerotic plaque and hepatic inflammation using histology and 2-deoxy-2-[18F]-fluoro- d -glucose (18F-FDG), a positron emission tomography tracer of inflammation, in a mouse model of hypercholesterolemia and type 2 diabetes. Methods Igf2/Ldlr−/−Apob100/100 mice were fed a high-fat diet (HFD) for 8 weeks and then randomly allocated to receive HFD (n = 14), or HFD with added linagliptin (n = 15) for additional 12 weeks. Five mice fed a chow diet were studied as an additional control. At the end of the study, glucose tolerance, aortic and liver uptake of 18F-FDG, and histology were studied. Results Mice in linagliptin and HFD groups had similar fasting glucose concentrations, but linagliptin improved glucose tolerance. Aortas of linagliptin and HFD groups showed advanced atherosclerotic plaques with no difference in the mean intima-to-media ratio or number of macrophages in the plaques. Autoradiography showed similar 18F-FDG uptake by atherosclerotic plaques in linagliptin and HFD groups (plaque-to-wall ratio: 1.7 ± 0.25 vs. 1.6 ± 0.21; p = 0.24). In the liver, linagliptin reduced the histologic inflammation score but had no effect on 18F-FDG uptake. Compared with chow diet, uptake of 18F-FDG was similar in the aorta, but higher in the liver after HFD. Conclusions Linagliptin therapy improved glucose tolerance and reduced hepatic inflammation but had no effect on plaque burden or atherosclerotic inflammation, as determined by histology and 18F-FDG uptake, in atherosclerotic mice with type 2 diabetes.

Aims The study aimed at investigating the use of guideline‐recommended diagnostic tools and medication in patients with heart failure (HF) in specialty care in Southwest Finland. We also compared the characteristics of the diagnosed and undiagnosed patients as well as laboratory tests, procedures, and treatments in everyday clinical practice. Methods and results Patients diagnosed with HF, cardiomyopathy, or hypertension‐induced heart disease (n = 20 878, primary cohort) or not diagnosed with HF but having a record of elevated N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) (>125 ng/L, n = 24 321, secondary cohort) were included in the study from the specialty care patient register of the Hospital District of Southwest Finland during the years 2005–2017. Among patients with an International Classification of Diseases, Tenth Revision (ICD‐10) code for HF, only 50% had ejection fraction (EF) data to be found by data mining from the electronic health records. Of these patients, 39% (n = 4042) had EF ≤ 40% [HF with reduced EF (HFrEF)] and 61% (n = 6347) had EF > 40%. Elevated NT‐proBNP together with EF > 40% narrowed down the number to 4590 patients, a population defined as HF with preserved EF (HFpEF) patients. HFpEF patients were further stratified into HF with mildly reduced EF (HFmrEF; EF 41–50%, n = 1468) and EF > 50% patients (n = 3122) to compare clinical characteristics. NT‐proBNP was higher within the HFrEF patients vs. HFpEF {4580 [inter‐quartile range (IQR): 2065–9765] vs. 2900 [2065–9765] ng/L, P 50% patients. In 70% (n = 17 156) of patients in the secondary cohort, the NT‐proBNP concentrations were >300 ng/L, median was 1090 (IQR 551–2558) ng/L and EF 58.4 ± 12.1% (n with EF available = 6845). Reduced EF was present in 6.8% of patients lacking HF diagnosis. Conclusions Half of the patients with ICD‐10 code for HF did not have EF data available after a visit at specialty care. In particular, the diagnosis of HFpEF seems challenging, reflected as an increase in procedures and laboratory test preceding diagnosis compared with those in HFrEF patients. Also, a large proportion of patients did not have HF diagnosis, yet they presented elevated NT‐proBNP concentrations and clinical characteristics resembling those of HFpEF patients.

Statins are effective drugs used to prevent and treat cardiovascular diseases but their effects in the absence of low density lipoprotein receptor (LDLR) and on the risk of diabetes are not yet well characterized. The aim of this study was to clarify systemic and pleiotropic effects of rosuvastatin on cardiovascular and diabetic phenotypes. IGF-II/LDLR-/-ApoB100/100 hypercholesterolemic prediabetic mice were used to test the effects of rosuvastatin on plasma glucose, insulin, lipids, atherosclerosis and liver steatosis. To get a more comprehensive view about changes in gene expression RNA-sequencing was done from the liver. Rosuvastatin significantly reduced plasma cholesterol in hypercholesterolemic mice in the absence of LDLR but had no effects on atherosclerosis at aortic sinus level or in coronary arteries. Rosuvastatin also significantly reduced liver steatosis without any harmful effects on glucose or insulin metabolism. RNA-sequencing showed relatively specific effects of rosuvastatin on genes involved in cholesterol metabolism together with a significant anti-inflammatory gene expression profile in the liver. In addition, significant changes were found in the expression of Perilipin 4 and 5 which are involved in lipid droplet formation in the liver. For the first time it could be shown that Tribbles proteins are affected by rosuvastatin treatment in the hyperlipidemic mice. Rosuvastatin had several positive effects on hypercholesterolemic mice showing early signs of diabetes, many of which are unrelated to cholesterol and lipoprotein metabolism. These results increase our understanding about the systemic and pleiotropic effects of rosuvastatin in the absence of LDLR expression.

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

VEGF-B gene therapy is a promising proangiogenic treatment for ischemic heart disease, but, unexpectedly, we found that high doses of VEGF-B promote ventricular arrhythmias (VAs). VEGF-B knockout, alpha myosin heavy-chain promoter (αMHC)-VEGF-B transgenic mice, and pigs transduced intramyocardially with adenoviral (Ad)VEGF- B186 were studied. Immunostaining showed a 2-fold increase in the number of nerves per field (76 vs. 39 in controls, p < 0.001) and an abnormal nerve distribution in the hypertrophic hearts of 11- to 20-month-old αMHC-VEGF-B mice. AdVEGF-B186 gene transfer (GT) led to local sprouting of nerve endings in pig myocardium (141 vs. 78 nerves per field in controls, p < 0.05). During dobutamine stress, 60% of the αMHC-VEGF-B hypertrophic mice had arrhythmias as compared to 7% in controls, and 20% of the AdVEGF-B186-transduced pigs and 100% of the combination of AdVEGF-B186- and AdsVEGFR-1-transduced pigs displayed VAs and even ventricular fibrillation. AdVEGF-B186 GT significantly increased the risk of sudden cardiac death in pigs when compared to any other GT with different VEGFs (hazard ratio, 500.5; 95% confidence interval [CI] 46.4–5,396.7; p < 0.0001). In gene expression analysis, VEGF-B induced the upregulation of Nr4a2, ATF6, and MANF in cardiomyocytes, molecules previously linked to nerve growth and differentiation. Thus, high AdVEGF-B186 overexpression induced nerve growth in the adult heart via a VEGFR-1 signaling-independent mechanism, leading to an increased risk of VA and sudden cardiac death.

Key points Transcriptional co-activator PGC-1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC-1α1 expression are not known. Transgenic PGC-1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling. The phenotype of the PGC-1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise-induced heart phenotype, but does not protect the heart against load-induced pathological hypertrophy. Transcriptional effects of PGC-1α1 show clear dose-dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC-1α1 expression levels. These results imply that the physiological role of PGC-1α1 is to promote a beneficial excitation–contraction coupling phenotype in the heart. Abstract The transcriptional coactivator PGC-1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC-1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC-1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC-1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation–contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC-1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus-induced PGC-1α1 expression, highlights PGC-1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose-dependent fashion. At low levels of overexpression PGC-1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC-1α1 are recruited at higher PGC-1α1 expression levels. Together these findings demonstrate that PGC-1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC-1α1 is to promote a beneficial EC coupling phenotype in the heart.

Gene therapy is a promising new treatment option for cardiac diseases. For finding the most suitable and safe vector for cardiac gene transfer, we delivered adenovirus (AdV), adeno-associated virus (AAV) and lentivirus (LeV) vectors into the mouse heart with sophisticated closed-chest echocardiography-guided intramyocardial injection method for comparing them with regards to transduction efficiency, myocardial damage, effects on the left ventricular function and electrocardiography (ECG). AdV had the highest transduction efficiency in cardiomyocytes followed by AAV2 and AAV9, and the lowest efficiency was seen with LeV. The local myocardial inflammation and fibrosis in the left ventricle (LV) was proportional to transduction efficiency. AdV caused LV dilatation and systolic dysfunction. Neither of the locally injected AAV serotypes impaired the LV systolic function, but AAV9 caused diastolic dysfunction to some extent. LeV did not affect the cardiac function. We also studied systemic delivery of AAV9, which led to transduction of cardiomyocytes throughout the myocardium. However, also diffuse fibrosis was present leading to significantly impaired LV systolic and diastolic function and pathological ECG changes. Compared with widely used AdV vector, AAV2, AAV9 and LeV were less effective in transducing cardiomyocytes but also less harmful. Local administration of AAV9 was safer and more efficient compared with systemic administration.

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

Vascular endothelial growth factor B (VEGF-B) is a potent mediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. In vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca2+ signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD60–70). These changes were partly caused by downregulation of sodium current (INa) due to reduced expression of Nav1.5. Furthermore, TG myocytes had alterations in voltage-gated K+ currents, namely decreased density of transient outward current (Ito) and total K+ current (Ipeak). At the level of transcription, these were accompanied by downregulation of Kv channel-interacting protein 2 (Kcnip2), a known modulatory subunit for Kv4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG., published version, peerReviewed

Adenoviruses are very efficient vectors for delivering therapeutic genes in preclinical and clinical trials. However, randomized controlled human trials have often been lacking clear clinically relevant results. We hypothesized that high lipid levels and specific lipoproteins could significantly decrease adenoviral transduction efficiency in vivo. Here we demonstrate that mice on a high fat diet have lower transgene expression compared to mice on a regular chow. In addition, on a high fat diet, ApoE−/− mice have much higher plasma transgene levels compared to LDLR-deficient mice. We also found that specific lipoprotein receptors play an important role in adenoviral transduction. These findings suggest that high plasma lipid levels, especially apoE-containing lipoproteins, reduce efficacy of adenoviral transduction in mice, which implies that high cholesterol levels in humans could be protective against viral infections and also lead to insufficient transgene expression in clinical trials using adenoviral vectors., published version, peerReviewed

Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20: 1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20: 1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modifi ed LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention.

Platelet-derived growth factors (PDGFs) are key regulators of mesenchymal cells in vertebrate development. To what extent PDGFs also exert beneficial homeostatic or reparative roles in adult organs, as opposed to adverse fibrogenic responses in pathology, are unclear. PDGF signaling plays critical roles during heart development, during which forced overexpression of PDGFs induces detrimental cardiac fibrosis; other studies have implicated PDGF signaling in post-infarct myocardial repair. Different PDGFs may exert different effects mediated through the two PDGF receptors (PDGFRα and PDGFRβ) in different cell types. Here, we assessed responses induced by five known PDGF isoforms in the adult mouse heart in the context of adenovirus vector-mediated inflammation. Our results show that different PDGFs have different, in some cases even opposing, effects. Strikingly, whereas the major PDGFRα agonists (PDGF-A and -C) decreased the amount of scar tissue and increased the numbers of PDGFRα-positive fibroblasts, PDGFRβ agonists either induced large scars with extensive inflammation (PDGF-B) or dampened the adenovirus-induced inflammation and produced a small and dense scar (PDGF-D). These results provide evidence for PDGF isoform-specific inflammation-modulating functions that may have therapeutic implications. They also illustrate a surprising complexity in the PDGF-mediated pathophysiological responses., published version, peerReviewed

Mechanisms of the transition from compensatory hypertrophy to heart failure are poorly understood and the roles of vascular endothelial growth factors (VEGFs) in this process have not been fully clarified. We determined the expression profile of VEGFs and relevant receptors during the progression of left ventricular hypertrophy (LVH). C57BL mice were exposed to transversal aortic constriction (TAC) and the outcome was studied at different time points (1 day, 2, 4, and 10 weeks). A clear compensatory phase (2 weeks after TAC) was seen with following heart failure (4 weeks after TAC). Interestingly, VEGF-C and VEGF-D as well as VEGF receptor-3 (VEGFR-3) were upregulated in the compensatory hypertrophy and VEGF-B was downregulated in the heart failure. After treatment with adeno-associated virus serotype 9 (AAV9)-VEGF-B(186) gene therapy in the compensatory phase for 4 weeks the function of the heart was preserved due to angiogenesis, inhibition of apoptosis, and promotion of cardiomyocyte proliferation. Also, the genetic programming towards fetal gene expression, a known phenomenon in heart failure, was partly reversed in AAV9-VEGF-B(186)-treated mice. We conclude that VEGF-C and VEGF-D are associated with the compensatory LVH and that AAV9-VEGF-B(186) gene transfer can rescue the function of the failing heart and postpone the transition towards heart failure.