Ubiquitin Proteasome System Role in Diabetes-Induced Cardiomyopathy.

This study investigated modifications to the ubiquitin proteasome system (UPS) in a mouse model of type 2 diabetes mellitus (T2DM) and their relationship to heart complications. db/db mice heart tissues were compared with WT mice tissues using RNA sequencing, qRT-PCR, and protein analysis to identify cardiac UPS modifications associated with diabetes. The findings unveiled a distinctive gene profile in the hearts of db/db mice with decreased levels of nppb mRNA and increased levels of Myh7, indicating potential cardiac dysfunction. The mRNA levels of USP18 (deubiquitinating enzyme), PSMB8, and PSMB9 (proteasome ß-subunits) were down-regulated in db/db mice, while the mRNA levels of RNF167 (E3 ligase) were increased. Corresponding LMP2 and LMP7 proteins were down-regulated in db/db mice, and RNF167 was elevated in Adult diabetic mice. The reduced expression of LMP2 and LMP7, along with increased RNF167 expression, may contribute to the future cardiac deterioration commonly observed in diabetes. This study enhances our understanding of UPS imbalances in the hearts of diabetic mice and raises questions about the interplay between the UPS and other cellular processes, such as autophagy. Further exploration in this area could provide valuable insights into the mechanisms underlying diabetic heart complications and potential therapeutic targets.

Effect of pharmacological heart failure drugs and gene therapy on Danon's cardiomyopathy.

Danon disease is a rare X-linked genetic disease resulting from LAMP2 mutations leading to defective lysosomal function. Heart failure is the main causes of morbidity and mortality. Mice with an LAMP2-exon-6-deletion (L2Δ6), develop cardiac hypertrophy followed by dilated cardiomyopathy, in association with accumulation of autophagosomes, fibrosis and oxidative stress. We investigated the effect of drugs used to treat heart failure and of LAMP2 gene therapy on the phenotype, molecular markers and ROS in LAMP2 cardiomyopathy. L2Δ6 mice were treated with Angiotensin II, Ramipril, Metoprolol or Spironolactone. Gene therapy was delivered by IP injection of Adeno-associated-virus (AAV9) -LAMP2 vector to neonates ("AAVLAMP2-Prevention"), or at 15 weeks of age ("AAVLAMP2-Treatment"). Angiotensin II markedly aggravated the cardiac phenotype. Ramipril and Spironolactone were effective in attenuating left ventricular hypertrophy and preserving the systolic function. Cardiac protection was associated with decreased autophagosome accumulation, reduced fibrosis and oxidative stress. Gene therapy effectively attenuated autophagosome accumulation and ROS in L2Δ6 hearts, lowering troponin release to nearly normal levels. AAVLAMP2-Prevention protected against systolic dysfunction and decreased hypertrophy. AAVLAMP2-Treatment prevented ventricular dilatation and dysfunction but had no effect on wall thickness. We conclude that RAAS inhibitors are highly effective against cardiomyopathy progression in an experimental mouse model of Danon's and shall be considered in human patients for this purpose until novel therapies become clinically available.

Silencing the Adipocytokine NOV: A Novel Approach to Reversing Oxidative Stress-Induced Cardiometabolic Dysfunction.

OBJECTIVE: NOV/CCN3 is an adipocytokine recently linked to obesity, insulin resistance, and cardiometabolic dysfunction. NOV is manufactured and secreted from adipose tissue, with blood levels highly correlated with BMI. NOV levels are increased in obesity and a myriad of inflammatory diseases. Elevated NOV levels cause oxidative stress by increasing free radicals, decreasing antioxidants, and decreasing heme oxygenase (HO-1) levels, resulting in decreased vascular function. Silencing NOV in NOV knockout mice improved insulin sensitivity. We wanted to study how suppressing NOV expression in an obese animal model affected pathways and processes related to obesity, inflammation, and cardiometabolic function. This is the first study to investigate the interaction of adipose tissue-specific NOV/CCN3 and cardiometabolic function. METHODS: We constructed a lentivirus containing the adiponectin-promoter-driven shNOV to examine the effect of NOV inhibition (shNOV) in adipose tissue on the heart of mice fed a high-fat diet. Mice were randomly divided into three groups (five per group): (1) lean (normal diet), (2) high-fat diet (HFD)+ sham virus, and (3) HFD + shNOV lentivirus. Blood pressure, tissue inflammation, and oxygen consumption were measured. Metabolic and mitochondrial markers were studied in fat and heart tissues. RESULTS: Mice fed an HFD developed adipocyte hypertrophy, fibrosis, inflammation, and decreased mitochondrial respiration. Inhibiting NOV expression in the adipose tissue of obese mice by shNOV increased mitochondrial markers for biogenesis (PGC-1α, the nuclear co-activator of HO-1) and functional integrity (FIS1) and insulin signaling (AKT). The upregulation of metabolic and mitochondrial markers was also evident in the hearts of the shNOV mice with the activation of mitophagy. Using RNA arrays, we identified a subgroup of genes that highly correlated with increased adipocyte mitochondrial autophagy in shNOV-treated mice. A heat map analysis in obese mice confirmed that the suppression of NOV overrides the genetic susceptibility of adiposity and the associated detrimental metabolic changes and correlates with the restoration of anti-inflammatory, thermogenic, and mitochondrial genes. CONCLUSION: Our novel findings demonstrate that inhibiting NOV expression improves adipose tissue function in a positive way in cardiometabolic function by inducing mitophagy and improving mitochondrial function by the upregulation of PGC-1α, the insulin sensitivity signaling protein. Inhibiting NOV expression increases PGC-1, a key component of cardiac bioenergetics, as well as key signaling components of metabolic change, resulting in improved glucose tolerance, improved mitochondrial function, and decreased inflammation. These metabolic changes resulted in increased oxygen consumption, decreased adipocyte size, and improved cardiac metabolism and vascular function at the structural level. The crosstalk of the adipose tissue-specific deletion of NOV/CCN3 improved cardiovascular function, representing a novel therapeutic strategy for obesity-related cardiometabolic dysfunction.

Autophagy guided interventions to modify the cardiac phenotype of Danon disease.

Danon disease is a lethal X-linked genetic syndrome resulting from radical mutations in the LAMP2 gene. LAMP2 protein deficiency results in defective lysosomal function, autophagy arrest and a multisystem disorder primarily involving the heart, skeletal muscle and the central nervous system. Cardiomyopathy is the main cause of morbidity and mortality. To investigate the mechanisms of and develop therapies for cardiac Danon disease we engineered a mouse model carrying an exon 6 deletion human mutation in LAMP2, which recapitulates the human cardiac disease phenotype. Mice develop cardiac hypertrophy followed by left ventricular dilatation and systolic dysfunction, in association with progressive fibrosis, oxidative stress, accumulation of autophagosomes and activation of proteasome. Stimulation of autophagy in Danon mice (by exercise training, caloric restriction, and rapamycin) aggravate the disease phenotype, promoting dilated cardiomyopathy. Inhibiting autophagy (by high fat diet or hydroxychloroquine) is better tolerated by Danon mice compared to wild type but is not curative. Inhibiting proteasome by Velcade was found to be highly toxic to Danon mice, suggesting that proteasome is activated to compensate for defective autophagy. In conclusion, activation of autophagy should be avoided in Danon patients. Since Danon's is a lifelong disease, we suggest that lifestyle interventions to decrease cardiac stress may be useful to slow progression of Danon's cardiomyopathy. While Danon mice better tolerate high fat diet and sedentary lifestyle, the benefit regarding cardiomyopathy in humans needs to be balanced against other health consequences of such interventions.

Adipocyte-Specific Expression of PGC1α Promotes Adipocyte Browning and Alleviates Obesity-Induced Metabolic Dysfunction in an HO-1-Dependent Fashion.

Recent studies suggest that PGC1-α plays a crucial role in mitochondrial and vascular function, yet the physiological significance of PGC1α and HO expression in adipose tissues in the context of obesity-linked vascular dysfunction remains unclear. We studied three groups of six-week-old C57BL/6J male mice: (1) mice fed a normal chow diet; (2) mice fed a high-fat diet (H.F.D.) for 28 weeks, and (3) mice fed a high-fat diet (H.F.D.) for 28 weeks, treated with adipose-specific overexpression of PGC-1α (transgenic-adipocyte-PGC-1α) at week 20, and continued on H.F.D. for weeks 20-28. R.N.A. arrays examined 88 genes involved in adipocyte proliferation and maturation. Blood pressure, tissue fibrosis, fasting glucose, and oxygen consumption were measured, as well as liver steatosis, and the expression levels of metabolic and mitochondrial markers. Obese mice exhibited a marked reduction of PGC1α and developed adipocyte hypertrophy, fibrosis, hepatic steatosis, and decreased mitochondrial respiration. Mice with adipose-specific overexpression of PGC1-α exhibited improvement in HO-1, mitochondrial biogenesis and respiration, with a decrease in fasting glucose, reduced blood pressure and fibrosis, and increased oxygen consumption. PGC-1α led to the upregulated expression of processes associated with the browning of fat tissue, including UCP1, FGF21, and pAMPK signaling, with a reduction in inflammatory adipokines, NOV/CCN3 expression, and TGFß. These changes required HO-1 expression. The R.N.A. array analysis identified subgroups of genes positively correlated with contributions to the browning of adipose tissue, all dependent on HO-1. Our observations reveal a positive impact of adipose-PGC1-α on distal organ systems, with beneficial effects on HO-1 levels, reversing obesity-linked cardiometabolic disturbances.

Simultaneous Noninvasive Detection and Therapy of Atherosclerosis Using HDL Coated Gold Nanorods.

Cardiovascular disease (CVD) is a major cause of death and disability worldwide. A real need exists in the development of new, improved therapeutic methods for treating CVD, while major advances in nanotechnology have opened new avenues in this field. In this paper, we report the use of gold nanoparticles (GNPs) coated with high-density lipoprotein (HDL) (GNP-HDL) for the simultaneous detection and therapy of unstable plaques. Based on the well-known HDL cardiovascular protection, by promoting the reverse cholesterol transport (RCT), injured rat carotids, as a model for unstable plaques, were injected with the GNP-HDL. Noninvasive detection of the plaques 24 h post the GNP injection was enabled using the diffusion reflection (DR) method, indicating that the GNP-HDL particles had accumulated in the injured site. Pathology and noninvasive CT measurements proved the recovery of the injured artery treated with the GNP-HDL. The DR of the GNP-HDL presented a simple and highly sensitive method at a low cost, resulting in simultaneous specific unstable plaque diagnosis and recovery.

Leptin modulates gene expression in the heart, cardiomyocytes and the adipose tissue thus mitigating LPS-induced damage.

Leptin is an adipokine of pleiotropic effects linked to energy metabolism, satiety, the immune response, and cardioprotection. We have recently shown that leptin causally conferred resistance to myocardial infarction-induced damage in transgenic αMUPA mice overexpressing leptin compared to their wild type (WT) ancestral mice FVB/N. Prompted by these findings, we have investigated here if leptin can counteract the inflammatory response triggered after LPS administration in tissues in vivo and in cardiomyocytes in culture. The results have shown that LPS upregulated in vivo and in vitro all genes examined here, both pro-inflammatory and antioxidant, as well as the leptin gene. Pretreating mice with leptin neutralizing antibodies further upregulated the expression of TNFα and IL-1ß in the adipose tissue of both mouse types, and in the αMUPA heart. The antibodies also increased the levels of serum markers for cell toxicity in both mouse types. These results indicate that under LPS, leptin actually reduced the levels of these inflammatory-related parameters. In addition, pretreatment with leptin antibodies reduced the levels of HIF-1α and VEGF mRNAs in the heart, indicating that under LPS leptin increased the levels of these mRNAs. In cardiomyocytes, pretreatment with exogenous leptin prior to LPS reduced the expression of both pro-inflammatory genes, enhanced the expression of the antioxidant genes HO-1, SOD2 and HIF-1α, and lowered ROS staining. In addition, results obtained with leptin antibodies and the SMLA leptin antagonist indicated that endogenous and exogenous leptin can inhibit leptin gene expression. Together, these findings have indicated that under LPS, leptin concomitantly downregulated pro-inflammatory genes, upregulated antioxidant genes, and lowered ROS levels. These results suggest that leptin can counteract inflammation in the heart and adipose tissue by modulating gene expression.

Expression of the SARS-CoV-2 receptorACE2 in human heart is associated with uncontrolled diabetes, obesity, and activation of the renin angiotensin system.

BACKGROUND: Diabetic and obese patients are at higher risk of severe disease and cardiac injury in corona virus 2 (SARS-CoV-2) infections. Cellular entry of SARS-CoV-2 is mainly via the angiotensin-converting enzyme 2 (ACE2) receptor, which is highly expressed in normal hearts. There is a disagreement regarding the effect of factors such as obesity and diabetes on ACE2 expression in the human heart and whether treatment with renin-angiotensin system inhibitors or anti-diabetic medications increases ACE2 expression and subsequently the susceptibility to infection. We designed this study to elucidate factors that control ACE2 expression in human serum, human heart biopsies, and mice. METHODS: Right atrial appendage biopsies were collected from 79 patients that underwent coronary artery bypass graft (CABG) surgery. We investigated the alteration in ACE2 mRNA and protein expression in heart tissue and serum. ACE2 expression was compared with clinical risk factors: diabetes, obesity and different anti-hypertensive or anti-diabetic therapies. WT or db/db mice were infused with Angiotensin II (ATII), treated with different anti-diabetic drugs (Metformin, GLP1A and SGLT2i) were also tested. RESULTS: ACE2 gene expression was increased in diabetic hearts compared to non-diabetic hearts and was positively correlated with glycosylated hemoglobin (HbA1c), body mass index (BMI), and activation of the renin angiotensin system (RAS), and negatively correlated with ejection fraction. ACE2 was not differentially expressed in patients who were on angiotensin converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARBs) prior to the operation. We found no correlation between plasma free ACE2 and cardiac tissue ACE2 expression. Transmembrane serine protease 2 (TMPRSS2), metalloprotease ADAM10 and ADAM17 that facilitate viral-ACE2 complex entry and degradation were increased in diabetic hearts. ACE2 expression in mice was increased with ATII infusion and attenuated following anti-diabetic drugs treatment. CONCLUSION: Patients with uncontrolled diabetes or obesity with RAS activation have higher ACE2 expressions therefore are at higher risk for severe infection. Since ACEi or ARBs show no effect on ACE2 expression in the heart further support their safety.

Leptin modulates gene expression in the heart and cardiomyocytes towards mitigating ischemia-induced damage.

Leptin, an adipocyte-derived satiety hormone, has been previously linked to cardioprotection. We have shown before that leptin conferred resistance to ischemic damage in the heart in long-lived transgenic αMUPA mice overexpressing leptin compared to the wild type (WT) FVB/N control mice. To better understand the contribution of leptin to the ischemic heart, we measured here the expression of genes encoding leptin and ischemia-related proteins in αMUPA and WT mice in the heart vs adipose tissue after MI. In addition, we investigated gene expression in neonatal rat cardiomyocytes under hypoxia in the absence and presence of exogenously added leptin or a leptin antagonist. We used real time RT-PCR and ELISA or Western blot assays to measure, respectively, mRNA and protein levels. The results have shown that circulating leptin levels and mRNA levels of leptin and heme oxygenase-1 (HO-1) in the heart were elevated in both mouse genotypes after 24 h myocardial infarction (MI), reaching higher values in αMUPA mice. In contrast, leptin gene expression in the adipose tissue was significantly increased only in WT mice, but reaching lower levels compared to the heart. Expression of the proinflammatory genes encoding TNFα and IL-1ß was also largely increased after MI in the heart in both mouse types, however reaching considerably lower levels in αMUPA mice indicating a mitigated inflammatory state. In cardiomyocytes, mRNA levels of all aforementioned genes as well as HIF-1α and SOD2 genes were elevated after hypoxia. Pretreatment with exogenous leptin largely reduced the mRNA levels of TNFα and IL-1ß after hypoxia, while enhancing expression of all other genes and reducing ROS levels. Pretreating the cells with a leptin antagonist increased solely the levels of leptin mRNA, suggesting a negative regulation of the hormone on the expression of its own gene. Overall, the results have shown that leptin affects expression of genes in cardiomyocytes under hypoxia in a manner that could mitigate inflammation and oxidative stress, suggesting a similar influence by endogenous leptin in αMUPA mice. Furthermore, leptin is likely to function in the ischemic murine heart more effectively in an autocrine compared to paracrine manner. These results suggest that leptin can reduce ischemic damage by modulating gene expression in the heart.

Cardiac Fibroblast-Induced Pluripotent Stem Cell-Derived Exosomes as a Potential Therapeutic Mean for Heart Failure.

The limited regenerative capacity of the injured myocardium leads to remodeling and often heart failure. Novel therapeutic approaches are essential. Induced pluripotent stem cells (iPSC) differentiated into cardiomyocytes are a potential future therapeutics. We hypothesized that organ-specific reprogramed fibroblasts may serve an advantageous source for future cardiomyocytes. Moreover, exosomes secreted from those cells may have a beneficial effect on cardiac differentiation and/or function. We compared RNA from different sources of human iPSC using chip gene expression. Protein expression was evaluated as well as exosome micro-RNA levels and their impact on embryoid bodies (EBs) differentiation. Statistical analysis identified 51 genes that were altered (p ≤ 0.05), and confirmed in the protein level, cardiac fibroblasts-iPSCs (CF-iPSCs) vs. dermal fibroblasts-iPSCs (DF-iPSCs). Several miRs were altered especially miR22, a key regulator of cardiac hypertrophy and remodeling. Lower expression of miR22 in CF-iPSCs vs. DF-iPSCs was observed. EBs treated with these exosomes exhibited more beating EBs p = 0.05. vs. control. We identify CF-iPSC and its exosomes as a potential source for cardiac recovery induction. The decrease in miR22 level points out that our CF-iPSC-exosomes are naïve of congestive heart cell memory, making them a potential biological source for future therapy for the injured heart.

Therapeutic approaches to diabetic cardiomyopathy: Targeting the antioxidant pathway.

The global epidemic of cardiovascular disease continues unabated and remains the leading cause of death both in the US and worldwide. We hereby summarize the available therapies for diabetes and cardiovascular disease in diabetics. Clearly, the current approaches to diabetic heart disease often target the manifestations and certain mediators but not the specific pathways leading to myocardial injury, remodeling and dysfunction. Better understanding of the molecular events determining the evolution of diabetic cardiomyopathy will provide insight into the development of specific and targeted therapies. Recent studies largely increased our understanding of the role of enhanced inflammatory response, ROS production, as well as the contribution of Cyp-P450-epoxygenase-derived epoxyeicosatrienoic acid (EET), Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1α (PGC-1α), Heme Oxygenase (HO)-1 and 20-HETE in pathophysiology and therapy of cardiovascular disease. PGC-1α increases production of the HO-1 which has a major role in protecting the heart against oxidative stress, microcirculation and mitochondrial dysfunction. This review describes the potential drugs and their downstream targets, PGC-1α and HO-1, as major loci for developing therapeutic approaches beside diet and lifestyle modification for the treatment and prevention of heart disease associated with obesity and diabetes.

The Peroxisome Proliferator-Activated Receptor-Gamma Coactivator-1α-Heme Oxygenase 1 Axis, a Powerful Antioxidative Pathway with Potential to Attenuate Diabetic Cardiomyopathy.

Significance: From studies of diabetic animal models, the downregulation of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α)-heme oxygenase 1 (HO-1) axis appears to be a crucial event in the development of obesity and diabetic cardiomyopathy (DCM). In this review, we discuss the role of metabolic and biochemical stressors in the rodent and human pathophysiology of DCM. A crucial contributor for many cardiac pathologies is excessive production of reactive oxygen species (ROS) pathologies, which lead to extensive cellular damage by impairing mitochondrial function and directly oxidizing DNA, proteins, and lipid membranes. We discuss the role of ROS production and inflammatory pathways with multiple contributing and confounding factors leading to DCM. Recent Advances: The relevant biochemical pathways that are critical to a therapeutic approach to treat DCM, specifically caloric restriction and its relation to the PGC-1α-HO-1 axis in the attenuation of DCM, are elucidated. Critical Issues: The increased prevalence of diabetes mellitus type 2, a major contributor to unique cardiomyopathy characterized by cardiomyocyte hypertrophy with no effective clinical treatment. This review highlights the role of mitochondrial dysfunction in the development of DCM and potential oxidative targets to attenuate oxidative stress and attenuate DCM. Future Directions: Targeting the PGC-1α-HO-1 axis is a promising approach to ameliorate DCM through improvement in mitochondrial function and antioxidant defenses. A pharmacological inducer to activate PGC-1α and HO-1 described in this review may be a promising therapeutic approach in the clinical setting.

The Role of Heme Oxygenase 1 in the Protective Effect of Caloric Restriction against Diabetic Cardiomyopathy.

Type 2 diabetes mellitus (DM2) leads to cardiomyopathy characterized by cardiomyocyte hypertrophy, followed by mitochondrial dysfunction and interstitial fibrosis, all of which are exacerbated by angiotensin II (AT). SIRT1 and its transcriptional coactivator target PGC-1α (peroxisome proliferator-activated receptor-γ coactivator), and heme oxygenase-1 (HO-1) modulates mitochondrial biogenesis and antioxidant protection. We have previously shown the beneficial effect of caloric restriction (CR) on diabetic cardiomyopathy through intracellular signaling pathways involving the SIRT1-PGC-1α axis. In the current study, we examined the role of HO-1 in diabetic cardiomyopathy in mice subjected to CR. METHODS: Cardiomyopathy was induced in obese diabetic (db/db) mice by AT infusion. Mice were either fed ad libitum or subjected to CR. In an in vitro study, the reactive oxygen species (ROS) level was determined in cardiomyocytes exposed to different glucose levels (7.5-33 mM). We examined the effects of Sn(tin)-mesoporphyrin (SnMP), which is an inhibitor of HO activity, the HO-1 inducer cobalt protoporphyrin (CoPP), and the SIRT1 inhibitor (EX-527) on diabetic cardiomyopathy. RESULTS: Diabetic mice had low levels of HO-1 and elevated levels of the oxidative marker malondialdehyde (MDA). CR attenuated left ventricular hypertrophy (LVH), increased HO-1 levels, and decreased MDA levels. SnMP abolished the protective effects of CR and caused pronounced LVH and cardiac metabolic dysfunction represented by suppressed levels of adiponectin, SIRT1, PPARγ, PGC-1α, and increased MDA. High glucose (33 mM) increased ROS in cultured cardiomyocytes, while SnMP reduced SIRT1, PGC-1α levels, and HO activity. Similarly, SIRT1 inhibition led to a reduction in PGC-1α and HO-1 levels. CoPP increased HO-1 protein levels and activity, SIRT1, and PGC-1α levels, and decreased ROS production, suggesting a positive feedback between SIRT1 and HO-1. CONCLUSION: These results establish a link between SIRT1, PGC-1α, and HO-1 signaling that leads to the attenuation of ROS production and diabetic cardiomyopathy. CoPP mimicked the beneficial effect of CR, while SnMP increased oxidative stress, aggravating cardiac hypertrophy. The data suggest that increasing HO-1 levels constitutes a novel therapeutic approach to protect the diabetic heart. Brief Summary: CR attenuates cardiomyopathy, and increases HO-1, SIRT activity, and PGC-1α protein levels in diabetic mice. High glucose reduces adiponectin, SIRT1, PGC1-1α, and HO-1 levels in cardiomyocytes, resulting in oxidative stress. The pharmacological activation of HO-1 activity mimics the effect of CR, while SnMP increased oxidative stress and cardiac hypertrophy. These data suggest the critical role of HO-1 in protecting the diabetic heart.

Protective Effect of TLR4 Ablation against Corneal Neovascularization following Chemical Burn in a Mouse Model.

PURPOSE: To determine whether Toll-like receptor 4 knockout protects mice from corneal neovascularization following chemical injury compared to wild-type (WT) mice. METHODS: A chemical burn (75% silver nitrate, 25% potassium nitrate) was created under anesthesia in the central right cornea of 32 WT and 31 Toll-like receptor 4 knockout mice. Corneal neovascularization was evaluated at 3, 4, 6, 8, 10, and 35 days after injury using digital photography, fluorescein angiography, gelatin perfusion with fluorescence vascular imaging, immunofluorescence staining, and molecular analysis. RESULTS: There was no significant between-group difference in relative corneal burn area at 10 days after injury (39.0 ± 2.4% vs. 38.8 ± 9.8%, respectively). Neovascularization was detected in all corneas in vivo and perfusion was detected by fluorescence vascular imaging, reaching maximum area on day 10. The relative area of neovascularization was significantly smaller in the knockout than the WT mice on days 6 (33.3 ± 4.2% vs. 46.8 ± 7.4%, respectively, p = 0.005) and 8 (36.6 ± 1.1% vs. 52.2 ± 6.4%, respectively, p = 0.027), although neovascularization was intensive in both groups. In line with the immunostaining findings of angiogenesis and inflammatory infiltration of damaged corneas, molecular analysis (performed on day 3) revealed elevated expression levels of angiogenesis-related genes (vascular endothelial growth factor, VEGFR2, VEGFR1) and inflammation-related genes (CD45 and TGFß1) in the WT mice. The knockout mice had higher TNF-α expression than the WT mice. CONCLUSION: In a mouse corneal chemical burn model, lack of Toll-like receptor 4 expression did not completely inhibit angiogenesis, but did have a relative effect to reduce neovascularization as compared to the WT.

Hyperlipidemic mice as a model for a real-time in vivo detection of atherosclerosis by gold nanorods-based diffusion reflection technique.

Atherosclerosis (AS), the leading cause of morbidity and mortality in cardiovascular disease, needs an early detection for treatment and prevention of fatal events. Here, for the first time, we applied gold nanorods (GNRs)-assisted diffusion reflection (DR), a noninvasive technique for in vivo detection of AS in a high-fat-diet-induced c57bl mouse model, which resembles the manifestation of AS in humans. DR simply detects the change in light reflection profile of tissue due to the accumulation of GNRs in the AS plaques and enables clear detection of AS lesions in carotid and femoral arteries of these hyperlipidemic mice. After 24 hours post-GNRs injection, DR showed the highest efficiency of AS detection. Moreover, the sensitivity of the DR method is much higher than computed tomography (CT) and is comparable to ex vivo high-resolution CT. Our results strongly suggest that the DR method can detect early atherosclerotic lesions in a sensitive and specific manner.

The Hyperpolarization-Activated Cyclic-Nucleotide-Gated Channel Blocker Ivabradine Does Not Prevent Arrhythmias in Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited, stressed-provoked ventricular arrhythmia. CPVT is treated by ß-adrenergic receptor blockers, Na+ channel inhibitors, sympathetic denervation, or by implanting a defibrillator. We showed recently that blockers of SK4 Ca2+-activated K+ channels depolarize the maximal diastolic potential, reduce the heart rate, and attenuate ventricular arrhythmias in CPVT. The aim of the present study was to examine whether the pacemaker channel inhibitor, ivabradine could demonstrate anti-arrhythmic properties in CPVT like other bradycardic agents used in this disease and to compare them with those of the SK4 channel blocker, TRAM-34. The effects of ivabradine were examined on the arrhythmic beating of human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) from CPVT patients, on sinoatrial node (SAN) calcium transients, and on ECG measurements obtained from transgenic mice model of CPVT. Ivabradine did neither prevent the arrhythmic pacing of hiPSC-CMs derived from CPVT patients, nor preclude the aberrant SAN calcium transients. In contrast to TRAM-34, ivabradine was unable to reduce in vivo the ventricular premature complexes and ventricular tachyarrhythmias in transgenic CPVT mice. In conclusion, ivabradine does not exhibit anti-arrhythmic properties in CPVT, which indicates that this blocker cannot be used as a plausible treatment for CPVT ventricular arrhythmias.

Structurally Simple, Readily Available Peptidomimetic 1-Benzyl-5-methyl-4-( n-octylamino)pyrimidin-2(1 H)-one Exhibited Efficient Cardioprotection in a Myocardial Ischemia (MI) Mouse Model.

TLR4, a member of the Toll-like receptor (TLR) family, serves as a pattern recognition receptor in the innate immune response to microbial pathogens. TLR4 also regulates the inflammatory reaction to ischemic injury in the heart. The TRIF-related adaptor molecule (TRAM) is an adapter that recruits the Toll/interleukin 1 receptor (TIR) domain, which contains adapter-inducing IFN-ß (TRIF), to activate TLR4, following TRIF-dependent cytokine gene transcription. On the basis of a known TRAM-derived decoy peptide, 10 of its peptidomimetics were synthesized. One of them, 1-benzyl-5-methyl-4-( n-octylamino)pyrimidin-2(1 H)-one (21), exhibited high potency and efficacy in vitro. In vitro results and in silico analysis provided evidence for the possible direct interaction of 21 with the TLR4 complex. Administered in mice, 21 was able to block the pathophysiological manifestation of MI, restoring the concomitant tissue damage, with a 100% survival rate. Thus, inhibition of TLR4-mediated inflammation in postischemic myocardium could be used as an approach for developing cardioprotective drugs.

PARP-1 inhibition protects the diabetic heart through activation of SIRT1-PGC-1α axis.

Type 2 diabetes mellitus (DM2) follows impaired glucose tolerance in obesity and is frequently associated with hypertension, causing adverse myocardial remodelling and leading to heart failure. The DNA bound protein PARP (poly ADP ribose) polymerase catalyses a post translational modification (polymerization of negatively charged ADP-ribose chains) of nuclear proteins. PARP-1 activation is NAD+ dependent and takes part in DNA repair and in chromatin remodelling and has a function in transcriptional regulation, intracellular trafficking and energy metabolism. PARP-1 is activated in diabetic cardiomyopathy. We hypothesized that PARP-1 inhibition in diabetic mice may protect cardiomyocytes from inflammation and ROS production. METHODS: Obese Leptin resistant (db/db) mice suffering from DM2, were treated with angiotensin II (AT) for 4 weeks to enhance the development of cardiomyopathy. Mice were concomitantly treated with the PARP-1 inhibitor INO1001. Neonatal cardiomyocytes exposed to high levels of glucose (33 mM) with or without AT were treated with INO1001. or with SIRT inhibitor (EX-527) in the presence of INO1001 were tested in-vitro. RESULTS: The in-vivo tests show that hearts from AT treated DM2 mice exhibited cardiac hypertrophy, fibrosis and an increase in the inflammatory marker TNFα. DM2 mice had an increased oxidative stress, concomitant with elevated PARP-1 activity and reduced Sirtuin-1 (SIRT1) expression. PARP-1 inhibition led to increased SIRT1 and Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) levels, attenuating oxidative stress, inflammation and fibrosis. In-vitro experiments demonstrated that inhibition of PARP-1 in cardiomyocytes exposed to high levels of glucose and AT led to a significant reduction in ROS (P < 0.01), which was abolished in the presence of the SIRT1 inhibitor together with increased protein expression of SIRT1 and PGC-1α. CONCLUSION: PARP1 inhibitor INO1001 attenuated cardiomyopathic features in diabetic mice through the activation of SIRT1 and its downstream antioxidant defence mechanisms. The results of this study suggest a pivotal role of PARP-1 inhibition in treating diabetic and AT-induced cardiomyopathy.

Correction to: Regulation of diabetic cardiomyopathy by caloric restriction is mediated by intracellular signaling pathways involving 'SIRT1 and PGC-1α'.

Unfortunately, after publication of this article [1], it was noticed that Table 1 contained errors introduced during the production process. In the WT + AT column, the FS value is 21 ± 7 and the Body Weight value is 25 ± 2. In the WT + AT + CR column, the FS value is 46 ± 14 and the Body Weight value is 19 ± 1. The original article has been updated to reflect this.