Possible retinotoxicity of long-term vardenafil treatment.

Phosphodiesterase (PDE) inhibitors - such as vardenafil - are used primarily for treating erectile dysfunction via increasing cyclic guanosine monophosphate (cGMP) levels. Recent studies have also demonstrated their significant cardioprotective effects in several diseases, including diabetes, upon long-term, continuous application. However, PDE inhibitors are not specific for PDE5 and also inhibit the retinal isoform. A sustained rise in cGMP in photoreceptors is known to be toxic; therefore, we hypothesized that long-term vardenafil treatment might result in retinotoxicity. The hypothesis was tested in a clinically relevant animal model of type 2 diabetes mellitus. Histological experiments were performed on lean and diabetic Zucker Diabetic Fatty rats. Half of the animals were treated with vardenafil for six months, and the retinal effects were evaluated. Vardenafil treatment alleviated rod outer segment degeneration but decreased rod numbers in some positions and induced changes in the interphotoreceptor matrix, even in control animals. Vardenafil treatment decreased total retinal thickness in the control and diabetic groups and reduced the number of nuclei in the outer nuclear layer. Müller cell activation was detectable even in the vardenafil-treated control animals, and vardenafil did not improve gliosis in the diabetic group. Vardenafil-treated animals showed complex retinal alterations with improvements in some parameters while deterioration in others. Our results point towards the retinotoxicity of vardenafil, even without diabetes, which raises doubts about the retinal safety of long-term continuous vardenafil administration. This effect needs to be considered when approving PDE inhibitors for alternative indications.

PCSK9 inhibition attenuates alcohol-associated neuronal oxidative stress and cellular injury.

Alcohol Use Disorder (AUD) is a persistent condition linked to neuroinflammation, neuronal oxidative stress, and neurodegenerative processes. While the inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has demonstrated effectiveness in reducing liver inflammation associated with alcohol, its impact on the brain remains largely unexplored. This study aimed to assess the effects of alirocumab, a monoclonal antibody targeting PCSK9 to lower systemic low-density lipoprotein cholesterol (LDL-C), on central nervous system (CNS) pathology in a rat model of chronic alcohol exposure. Alirocumab (50 mg/kg) or vehicle was administered weekly for six weeks in 32 male rats subjected to a 35 % ethanol liquid diet or a control liquid diet (n = 8 per group). The study evaluated PCSK9 expression, LDL receptor (LDLR) expression, oxidative stress, and neuroinflammatory markers in brain tissues. Chronic ethanol exposure increased PCSK9 expression in the brain, while alirocumab treatment significantly upregulated neuronal LDLR and reduced oxidative stress in neurons and brain vasculature (3-NT, p22phox). Alirocumab also mitigated ethanol-induced microglia recruitment in the cortex and hippocampus (Iba1). Additionally, alirocumab decreased the expression of pro-inflammatory cytokines and chemokines (TNF, CCL2, CXCL3) in whole brain tissue and attenuated the upregulation of adhesion molecules in brain vasculature (ICAM1, VCAM1, eSelectin). This study presents novel evidence that alirocumab diminishes oxidative stress and modifies neuroimmune interactions in the brain elicited by chronic ethanol exposure. Further investigation is needed to elucidate the mechanisms by which PCSK9 signaling influences the brain in the context of chronic ethanol exposure.

Interplay of Liver-Heart Inflammatory Axis and Cannabinoid 2 Receptor Signaling in an Experimental Model of Hepatic Cardiomyopathy.

BACKGROUND AND AIMS: Hepatic cardiomyopathy, a special type of heart failure, develops in up to 50% of patients with cirrhosis and is a major determinant of survival. However, there is no reliable model of hepatic cardiomyopathy in mice. We aimed to characterize the detailed hemodynamics of mice with bile duct ligation (BDL)-induced liver fibrosis, by monitoring echocardiography and intracardiac pressure-volume relationships and myocardial structural alterations. Treatment of mice with a selective cannabinoid-2 receptor (CB2 -R) agonist, known to attenuate inflammation and fibrosis, was used to explore the impact of liver inflammation and fibrosis on cardiac function. APPROACH AND RESULTS: BDL induced massive inflammation (increased leukocyte infiltration, inflammatory cytokines, and chemokines), oxidative stress, microvascular dysfunction, and fibrosis in the liver. These pathological changes were accompanied by impaired diastolic, systolic, and macrovascular functions; cardiac inflammation (increased macrophage inflammatory protein 1, interleukin-1, P-selectin, cluster of differentiation 45-positive cells); and oxidative stress (increased malondialdehyde, 3-nitrotyrosine, and nicotinamide adenine dinucleotide phosphate oxidases). CB2 -R up-regulation was observed in both livers and hearts of mice exposed to BDL. CB2 -R activation markedly improved hepatic inflammation, impaired microcirculation, and fibrosis. CB2 -R activation also decreased serum tumor necrosis factor-alpha levels and improved cardiac dysfunction, myocardial inflammation, and oxidative stress, underlining the importance of inflammatory mediators in the pathology of hepatic cardiomyopathy. CONCLUSIONS: We propose BDL-induced cardiomyopathy in mice as a model for hepatic/cirrhotic cardiomyopathy. This cardiomyopathy, similar to cirrhotic cardiomyopathy in humans, is characterized by systemic hypotension and impaired macrovascular and microvascular function accompanied by both systolic and diastolic dysfunction. Our results indicate that the liver-heart inflammatory axis has a pivotal pathophysiological role in the development of hepatic cardiomyopathy. Thus, controlling liver and/or myocardial inflammation (e.g., with selective CB2 -R agonists) may delay or prevent the development of cardiomyopathy in severe liver disease.

Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is a clinical syndrome defined by liver failure on pre-existing chronic liver disease. It is often associated with bacterial infection and high short-term mortality. Experimental models that fully reproduce ACLF are lacking, so too are effective pharmacological therapies for this condition. METHODS: To mimic ACLF conditions, we developed a severe liver injury model by combining chronic injury (chronic carbon tetrachloride [CCl4] injection), acute hepatic insult (injection of a double dose of CCl4), and bacterial infection (intraperitoneal injection of bacteria). Serum and liver samples from patients with ACLF or acute drug-induced liver injury (DILI) were used. Liver injury and regeneration were assessed to ascertain the potential benefits of interleukin-22 (IL-22Fc) administration. RESULTS: This severe liver injury model recapitulated some of the key features of clinical ACLF, including acute-on-chronic liver injury, bacterial infection, multi-organ injury, and high mortality. Liver regeneration in this model was severely impaired because of a shift from the activation of the pro-regenerative IL-6/STAT3 pathway to the anti-regenerative IFN-γ/STAT1 pathway. The impaired IL-6/STAT3 activation was due to the inability of Kupffer cells to produce IL-6; whereas the enhanced STAT1 activation was due to a strong innate immune response and subsequent production of IFN-γ. Compared to patients with DILI, patients with ACLF had higher levels of IFN-γ but lower liver regeneration. IL-22Fc treatment improved survival in ACLF mice by reversing the STAT1/STAT3 pathway imbalance and enhancing expression of many antibacterial genes in a manner involving the anti-apoptotic protein BCL2. CONCLUSIONS: Acute-on-chronic liver injury or bacterial infection is associated with impaired liver regeneration due to a shift from a pro-regenerative to an anti-regenerative pathway. IL-22Fc therapy reverses this shift and attenuates bacterial infection, thus IL-22Fc may have therapeutic potential for ACLF treatment. LAY SUMMARY: A mouse model combining chronic liver injury, acute hepatic insult, and bacterial infection recapitulates some of the key features of acute-on-chronic liver failure (ACLF) in patients. Both fibrosis and bacterial infection contribute to the impaired regenerative capacity of the liver in patients with ACLF. Herein, we show that IL-22Fc therapy improves ACLF by reprogramming impaired regenerative pathways and attenuating bacterial infection. Thus, it may have therapeutic potential for patients with ACLF.

Disruption of Renal Arginine Metabolism Promotes Kidney Injury in Hepatorenal Syndrome in Mice.

Tubular dysfunction is an important feature of renal injury in hepatorenal syndrome (HRS) in patients with end-stage liver disease. The pathogenesis of kidney injury in HRS is elusive, and there are no clinically relevant rodent models of HRS. We investigated the renal consequences of bile duct ligation (BDL)-induced hepatic and renal injury in mice in vivo by using biochemical assays, real-time polymerase chain reaction (PCR), Western blot, mass spectrometry, histology, and electron microscopy. BDL resulted in time-dependent hepatic injury and hyperammonemia which were paralleled by tubular dilation and tubulointerstitial nephritis with marked upregulation of lipocalin-2, kidney injury molecule 1 (KIM-1) and osteopontin. Renal injury was associated with dramatically impaired microvascular flow and decreased endothelial nitric oxide synthase (eNOS) activity. Gene expression analyses signified proximal tubular epithelial injury, tissue hypoxia, inflammation, and activation of the fibrotic gene program. Marked changes in renal arginine metabolism (upregulation of arginase-2 and downregulation of argininosuccinate synthase 1), resulted in decreased circulating arginine levels. Arginase-2 knockout mice were partially protected from BDL-induced renal injury and had less impairment in microvascular function. In human-cultured proximal tubular epithelial cells hyperammonemia per se induced upregulation of arginase-2 and markers of tubular cell injury. CONCLUSION: We propose that hyperammonemia may contribute to impaired renal arginine metabolism, leading to decreased eNOS activity, impaired microcirculation, tubular cell death, tubulointerstitial nephritis and fibrosis. Genetic deletion of arginase-2 partially restores microcirculation and thereby alleviates tubular injury. We also demonstrate that BDL in mice is an excellent, clinically relevant model to study the renal consequences of HRS. (Hepatology 2018; 00:000-000).

Comparison of speckle-tracking echocardiography with invasive hemodynamics for the detection of characteristic cardiac dysfunction in type-1 and type-2 diabetic rat models.

BACKGROUND: Measurement of systolic and diastolic function in animal models is challenging by conventional non-invasive methods. Therefore, we aimed at comparing speckle-tracking echocardiography (STE)-derived parameters to the indices of left ventricular (LV) pressure-volume (PV) analysis to detect cardiac dysfunction in rat models of type-1 (T1DM) and type-2 (T2DM) diabetes mellitus. METHODS: Rat models of T1DM (induced by 60 mg/kg streptozotocin, n = 8) and T2DM (32-week-old Zucker Diabetic Fatty rats, n = 7) and corresponding control animals (n = 5 and n = 8, respectively) were compared. Echocardiography and LV PV analysis were performed. LV short-axis recordings were used for STE analysis. Global circumferential strain, peak strain rate values in systole (SrS), isovolumic relaxation (SrIVR) and early diastole (SrE) were measured. LV contractility, active relaxation and stiffness were measured by PV analysis. RESULTS: In T1DM, contractility and active relaxation were deteriorated to a greater extent compared to T2DM. In contrast, diastolic stiffness was impaired in T2DM. Correspondingly, STE described more severe systolic dysfunction in T1DM. Among diastolic STE parameters, SrIVR was more decreased in T1DM, however, SrE was more reduced in T2DM. In T1DM, SrS correlated with contractility, SrIVR with active relaxation, while in T2DM SrE was related to cardiac stiffness, cardiomyocyte diameter and fibrosis. CONCLUSIONS: Strain and strain rate parameters can be valuable and feasible measures to describe the dynamic changes in contractility, active relaxation and LV stiffness in animal models of T1DM and T2DM. STE corresponds to PV analysis and also correlates with markers of histological myocardial remodeling.

Cannabidiol Limits T Cell-Mediated Chronic Autoimmune Myocarditis: Implications to Autoimmune Disorders and Organ Transplantation.

Myocarditis is a major cause of heart failure and sudden cardiac death in young adults and adolescents. Many cases of myocarditis are associated with autoimmune processes in which cardiac myosin is a major autoantigen. Conventional immunosuppressive therapies often provide unsatisfactory results and are associated with adverse toxicities during the treatment of autoimmune myocarditis. Cannabidiol (CBD) is a nonpsychoactive constituent of marijuana that exerts antiinflammatory effects independent of classical cannabinoid receptors. Recently, 80 clinical trials have investigated the effects of CBD in various diseases from inflammatory bowel disease to graft versus host disease. CBD-based formulations are used for the management of multiple sclerosis in numerous countries, and CBD also received U.S. Food and Drug Administration approval for the treatment of refractory childhood epilepsy and glioblastoma multiforme. Herein, using a well-established mouse model of experimental autoimmune myocarditis (EAM) induced by immunization with cardiac myosin emmulsified in adjuvant resulting in T cell-mediated inflammation, cardiomyocyte cell death, fibrosis and myocardial dysfunction, we studied the potential beneficial effects of CBD. EAM was characterized by marked myocardial T-cell infiltration, profound inflammatory response and fibrosis (measured by quantitative real-time polymerase chain reaction, histology and immunohistochemistry analyses) accompanied by marked attenuation of both systolic and diastolic cardiac functions measured with a pressure-volume conductance catheter technique. Chronic treatment with CBD largely attenuated the CD3+ and CD4+ T cell-mediated inflammatory response and injury, myocardial fibrosis and cardiac dysfunction in mice. In conclusion, CBD may represent a promising novel treatment for managing autoimmune myocarditis and possibly other autoimmune disorders and organ transplantation.

Chronic plus binge ethanol feeding induces myocardial oxidative stress, mitochondrial and cardiovascular dysfunction, and steatosis.

Alcoholic cardiomyopathy in humans develops in response to chronic excessive alcohol consumption; however, good models of alcohol-induced cardiomyopathy in mice are lacking. Herein we describe mouse models of alcoholic cardiomyopathies induced by chronic and binge ethanol (EtOH) feeding and characterize detailed hemodynamic alterations, mitochondrial function, and redox signaling in these models. Mice were fed a liquid diet containing 5% EtOH for 10, 20, and 40 days (d) combined with single or multiple EtOH binges (5 g/kg body wt). Isocalorically pair-fed mice served as controls. Left ventricular (LV) function and morphology were assessed by invasive pressure-volume conductance approach and by echocardiography. Mitochondrial complex (I, II, IV) activities, 3-nitrotyrosine (3-NT) levels, gene expression of markers of oxidative stress (gp91phox, p47phox), mitochondrial biogenesis (PGC1α, peroxisome proliferator-activated receptor α), and fibrosis were examined. Cardiac steatosis and fibrosis were investigated by histological/immunohistochemical methods. Chronic and binge EtOH feeding (already in 10 days EtOH plus single binge group) was characterized by contractile dysfunction (decreased slope of end-systolic pressure-volume relationship and preload recruitable stroke work), impaired relaxation (decreased time constant of LV pressure decay and maximal slope of systolic pressure decrement), and vascular dysfunction (impaired arterial elastance and lower total peripheral resistance). This was accompanied by enhanced myocardial oxidative/nitrative stress (3-NT; gp91phox; p47phox; angiotensin II receptor, type 1a) and deterioration of mitochondrial complex I, II, IV activities and mitochondrial biogenesis, excessive cardiac steatosis, and higher mortality. Collectively, chronic plus binge EtOH feeding in mice leads to alcohol-induced cardiomyopathies (National Institute on Alcohol Abuse and Alcoholism models) characterized by increased myocardial oxidative/nitrative stress, impaired mitochondrial function and biogenesis, and enhanced cardiac steatosis.

Myocardial reverse remodeling after pressure unloading is associated with maintained cardiac mechanoenergetics in a rat model of left ventricular hypertrophy.

Pressure unloading represents the only effective therapy in increased afterload-induced left ventricular hypertrophy (LVH) as it leads to myocardial reverse remodeling (reduction of increased left ventricular mass, attenuated myocardial fibrosis) and preserved cardiac function. However, the effect of myocardial reverse remodeling on cardiac mechanoenergetics has not been elucidated. Therefore, we aimed to provide a detailed hemodynamic characterization in a rat model of LVH undergoing pressure unloading. Pressure overload was induced in Sprague-Dawley rats by abdominal aortic banding for 6 (AB 6th wk) or 12 wk (AB 12th wk). Sham-operated animals served as controls. Aortic debanding procedure was performed after the 6th experimental week (debanded 12th wk) to investigate the regression of LVH. Pressure unloading resulted in significant reduction of LVH (heart weight-to-tibial length ratio: 0.38 ± 0.01 vs. 0.58 ± 0.02 g/mm, cardiomyocyte diameter: 18.3 ± 0.1 vs. 24.1 ± 0.8 µm debanded 12th wk vs. AB 12th wk, P < 0.05), attenuated the extracellular matrix remodeling (Masson's score: 1.37 ± 0.13 vs. 1.73 ± 0.10, debanded 12th wk vs. AB 12th wk, P < 0.05), provided protection against the diastolic dysfunction, and reversed the maladaptive contractility augmentation (slope of end-systolic pressure-volume relationship: 1.39 ± 0.24 vs. 2.04 ± 0.09 mmHg/µl, P < 0.05 debanded 12th wk vs. AB 6th wk, P < 0.05). In addition, myocardial reverse remodeling was also associated with preserved ventriculoarterial coupling and increased mechanical efficiency (50.6 ± 2.8 vs. 38.9 ± 2.5%, debanded 12th wk vs. AB 12th wk, P < 0.05), indicating a complete functional and mechanoenergetic recovery. According to our best knowledge, this is the first study demonstrating that the regression of LVH is accompanied by maintained cardiac mechanoenergetics.

Physiological and pathological left ventricular hypertrophy of comparable degree is associated with characteristic differences of in vivo hemodynamics.

Left ventricular (LV) hypertrophy is a physiological or pathological response of LV myocardium to increased cardiac load. We aimed at investigating and comparing hemodynamic alterations in well-established rat models of physiological hypertrophy (PhyH) and pathological hypertrophy (PaH) by using LV pressure-volume (P-V) analysis. PhyH and PaH were induced in rats by swim training and by abdominal aortic banding, respectively. Morphology of the heart was investigated by echocardiography. Characterization of cardiac function was completed by LV P-V analysis. In addition, histological and molecular biological measurements were performed. Echocardiography revealed myocardial hypertrophy of similar degree in both models, which was confirmed by post-mortem heart weight data. In aortic-banded rats we detected subendocardial fibrosis. Reactivation of fetal gene program could be observed only in the PaH model. PhyH was associated with increased stroke volume, whereas unaltered stroke volume was detected in PaH along with markedly elevated end-systolic pressure values. Sensitive indexes of LV contractility were increased in both models, in parallel with the degree of hypertrophy. Active relaxation was ameliorated in athlete's heart, whereas it showed marked impairment in PaH. Mechanical efficiency and ventriculo-arterial coupling were improved in PhyH, whereas they remained unchanged in PaH. Myocardial gene expression of mitochondrial regulators showed marked differences between PaH and PhyH. We provided the first comparative hemodynamic characterization of PhyH and PaH in relevant rodent models. Increased LV contractility could be observed in both types of LV hypertrophy; characteristic distinction was detected in diastolic function (active relaxation) and mechanoenergetics (mechanical efficiency), which might be explained by mitochondrial differences.

Diastolic dysfunction in prediabetic male rats: Role of mitochondrial oxidative stress.

Although incidence and prevalence of prediabetes are increasing, little is known about its cardiac effects. Therefore, our aim was to investigate the effect of prediabetes on cardiac function and to characterize parameters and pathways associated with deteriorated cardiac performance. Long-Evans rats were fed with either control or high-fat chow for 21 wk and treated with a single low dose (20 mg/kg) of streptozotocin at week 4 High-fat and streptozotocin treatment induced prediabetes as characterized by slightly elevated fasting blood glucose, impaired glucose and insulin tolerance, increased visceral adipose tissue and plasma leptin levels, as well as sensory neuropathy. In prediabetic animals, a mild diastolic dysfunction was observed, the number of myocardial lipid droplets increased, and left ventricular mass and wall thickness were elevated; however, no molecular sign of fibrosis or cardiac hypertrophy was shown. In prediabetes, production of reactive oxygen species was elevated in subsarcolemmal mitochondria. Expression of mitofusin-2 was increased, while the phosphorylation of phospholamban and expression of Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3, a marker of mitophagy) decreased. However, expression of other markers of cardiac auto- and mitophagy, mitochondrial dynamics, inflammation, heat shock proteins, Ca2+/calmodulin-dependent protein kinase II, mammalian target of rapamycin, or apoptotic pathways were unchanged in prediabetes. This is the first comprehensive analysis of cardiac effects of prediabetes indicating that mild diastolic dysfunction and cardiac hypertrophy are multifactorial phenomena that are associated with early changes in mitophagy, cardiac lipid accumulation, and elevated oxidative stress and that prediabetes-induced oxidative stress originates from the subsarcolemmal mitochondria.

Strain and strain rate by speckle-tracking echocardiography correlate with pressure-volume loop-derived contractility indices in a rat model of athlete's heart.

Contractile function is considered to be precisely measurable only by invasive hemodynamics. We aimed to correlate strain values measured by speckle-tracking echocardiography (STE) with sensitive contractility parameters of pressure-volume (P-V) analysis in a rat model of exercise-induced left ventricular (LV) hypertrophy. LV hypertrophy was induced in rats by swim training and was compared with untrained controls. Echocardiography was performed using a 13-MHz linear transducer to obtain LV long- and short-axis recordings for STE analysis (GE EchoPAC). Global longitudinal (GLS) and circumferential strain (GCS) and longitudinal (LSr) and circumferential systolic strain rate (CSr) were measured. LV P-V analysis was performed using a pressure-conductance microcatheter, and load-independent contractility indices [slope of the end-systolic P-V relationship (ESPVR), preload recruitable stroke work (PRSW), and maximal dP/dt-end-diastolic volume relationship (dP/dtmax-EDV)] were calculated. Trained rats had increased LV mass index (trained vs. control; 2.76 ± 0.07 vs. 2.14 ± 0.05 g/kg, P < 0.001). P-V loop-derived contractility parameters were significantly improved in the trained group (ESPVR: 3.58 ± 0.22 vs. 2.51 ± 0.11 mmHg/µl; PRSW: 131 ± 4 vs. 104 ± 2 mmHg, P < 0.01). Strain and strain rate parameters were also supernormal in trained rats (GLS: -18.8 ± 0.3 vs. -15.8 ± 0.4%; LSr: -5.0 ± 0.2 vs. -4.1 ± 0.1 Hz; GCS: -18.9 ± 0.8 vs. -14.9 ± 0.6%; CSr: -4.9 ± 0.2 vs. -3.8 ± 0.2 Hz, P < 0.01). ESPVR correlated with GLS (r = -0.71) and LSr (r = -0.53) and robustly with GCS (r = -0.83) and CSr (r = -0.75, all P < 0.05). PRSW was strongly related to GLS (r = -0.64) and LSr (r = -0.71, both P < 0.01). STE can be a feasible and useful method for animal experiments. In our rat model, strain and strain rate parameters closely reflected the improvement in intrinsic contractile function induced by exercise training.

The soluble guanylate cyclase activator cinaciguat prevents cardiac dysfunction in a rat model of type-1 diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) leads to the development of diabetic cardiomyopathy, which is associated with altered nitric oxide (NO)--soluble guanylate cyclase (sGC)--cyclic guanosine monophosphate (cGMP) signalling. Cardioprotective effects of elevated intracellular cGMP-levels have been described in different heart diseases. In the current study we aimed at investigating the effects of pharmacological activation of sGC in diabetic cardiomyopathy. METHODS: Type-1 DM was induced in rats by streptozotocin. Animals were treated either with the sGC activator cinaciguat (10 mg/kg/day) or with placebo orally for 8 weeks. Left ventricular (LV) pressure-volume (P-V) analysis was used to assess cardiac performance. Additionally, gene expression (qRT-PCR) and protein expression analysis (western blot) were performed. Cardiac structure, markers of fibrotic remodelling and DNA damage were examined by histology, immunohistochemistry and TUNEL assay, respectively. RESULTS: DM was associated with deteriorated cGMP signalling in the myocardium (elevated phosphodiesterase-5 expression, lower cGMP-level and impaired PKG activity). Cardiomyocyte hypertrophy, fibrotic remodelling and DNA fragmentation were present in DM that was associated with impaired LV contractility (preload recruitable stroke work (PRSW): 49.5 ± 3.3 vs. 83.0 ± 5.5 mmHg, P < 0.05) and diastolic function (time constant of LV pressure decay (Tau): 17.3 ± 0.8 vs. 10.3 ± 0.3 ms, P < 0.05). Cinaciguat treatment effectively prevented DM related molecular, histological alterations and significantly improved systolic (PRSW: 66.8 ± 3.6 mmHg) and diastolic (Tau: 14.9 ± 0.6 ms) function. CONCLUSIONS: Cinaciguat prevented structural, molecular alterations and improved cardiac performance of the diabetic heart. Pharmacological activation of sGC might represent a new therapy approach for diabetic cardiomyopathy.

Rat model of exercise-induced cardiac hypertrophy: hemodynamic characterization using left ventricular pressure-volume analysis.

Long-term exercise training is associated with characteristic structural and functional changes of the myocardium, termed athlete's heart. Several research groups investigated exercise training-induced left ventricular (LV) hypertrophy in animal models; however, only sporadic data exist about detailed hemodynamics. We aimed to provide functional characterization of exercise-induced cardiac hypertrophy in a rat model using the in vivo method of LV pressure-volume (P-V) analysis. After inducing LV hypertrophy by swim training, we assessed LV morphometry by echocardiography and performed LV P-V analysis using a pressure-conductance microcatheter to investigate in vivo cardiac function. Echocardiography showed LV hypertrophy (LV mass index: 2.41 ± 0.09 vs. 2.03 ± 0.08 g/kg, P < 0.01), which was confirmed by heart weight data and histomorphometry. Invasive hemodynamic measurements showed unaltered heart rate, arterial pressure, and LV end-diastolic volume along with decreased LV end-systolic volume, thus increased stroke volume and ejection fraction (73.7 ± 0.8 vs. 64.1 ± 1.5%, P < 0.01) in trained versus untrained control rats. The P-V loop-derived sensitive, load-independent contractility indexes, such as slope of end-systolic P-V relationship or preload recruitable stroke work (77.0 ± 6.8 vs. 54.3 ± 4.8 mmHg, P = 0.01) were found to be significantly increased. The observed improvement of ventriculoarterial coupling (0.37 ± 0.02 vs. 0.65 ± 0.08, P < 0.01), along with increased LV stroke work and mechanical efficiency, reflects improved mechanoenergetics of exercise-induced cardiac hypertrophy. Despite the significant hypertrophy, we observed unaltered LV stiffness (slope of end-diastolic P-V relationship: 0.043 ± 0.007 vs. 0.040 ± 0.006 mmHg/µl) and improved LV active relaxation (τ: 10.1 ± 0.6 vs. 11.9 ± 0.2 ms, P < 0.01). According to our knowledge, this is the first study that provides characterization of functional changes and hemodynamic relations in exercise-induced cardiac hypertrophy.

Data-driven transcriptomics analysis identifies PCSK9 as a novel key regulator in liver aging.

The liver, as a crucial metabolic organ, undergoes significant pathological changes during the aging process, which can have a profound impact on overall health. To gain a comprehensive understanding of these alterations, we employed data-driven approaches, along with biochemical methods, histology, and immunohistochemistry techniques, to systematically investigate the effects of aging on the liver. Our study utilized a well-established rat aging model provided by the National Institute of Aging. Systems biology approaches were used to analyze genome-wide transcriptomics data from liver samples obtained from young (4-5 months old) and aging (20-21 months old) Fischer 344 rats. Our findings revealed pathological changes occurring in various essential biological processes in aging livers. These included mitochondrial dysfunction, increased oxidative/nitrative stress, decreased NAD + content, impaired amino acid and protein synthesis, heightened inflammation, disrupted lipid metabolism, enhanced apoptosis, senescence, and fibrosis. These results were validated using independent datasets from both human and rat aging studies. Furthermore, by employing co-expression network analysis, we identified novel driver genes responsible for liver aging, confirmed our findings in human aging subjects, and pointed out the cellular localization of the driver genes using single-cell RNA-sequencing human data. Our study led to the discovery and validation of a liver-specific gene, proprotein convertase subtilisin/kexin type 9 (PCSK9), as a potential therapeutic target for mitigating the pathological processes associated with aging in the liver. This finding envisions new possibilities for developing interventions aimed to improve liver health during the aging process.

PCSK9, A Promising Novel Target for Age-Related Cardiovascular Dysfunction.

Cardiovascular diseases (CVDs) are the leading cause of death among elderly people. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important regulator of cholesterol metabolism. Herein, we investigated the role of PCSK9 in age-related CVD. Both in humans and rats, blood PCSK9 level correlated positively with increasing age and the development of cardiovascular dysfunction. Age-related fatty degeneration of liver tissue positively correlated with serum PCSK9 levels in the rat model, while development of age-related nonalcoholic fatty liver disease correlated with cardiovascular functional impairment. Network analysis identified PCSK9 as an important factor in age-associated lipid alterations and it correlated positively with intima-media thickness, a clinical parameter of CVD risk. PCSK9 inhibition with alirocumab effectively reduced the CVD progression in aging rats, suggesting that PCSK9 plays an important role in cardiovascular aging.

Cannabinoid receptor 2 activation alleviates diabetes-induced cardiac dysfunction, inflammation, oxidative stress, and fibrosis.

Diabetes mellitus promotes accelerated cardiovascular aging and inflammation, which in turn facilitate the development of cardiomyopathy/heart failure. High glucose-induced oxidative/nitrative stress, activation of various pro-inflammatory, and cell death pathways are critical in the initiation and progression of the changes culminating in diabetic cardiomyopathy. Cannabinoid 2 receptor (CB2R) activation in inflammatory cells and activated endothelium attenuates the pathological changes associated with atherosclerosis, myocardial infarction, stroke, and hepatic cardiomyopathy. In this study, we explored the role of CB2R signaling in myocardial dysfunction, oxidative/nitrative stress, inflammation, cell death, remodeling, and fibrosis associated with diabetic cardiomyopathy in type 1 diabetic mice. Control human heart left ventricles and atrial appendages, similarly to mouse hearts, had negligible CB2R expression determine by RNA sequencing or real-time RT-PCR. Diabetic cardiomyopathy was characterized by impaired diastolic and systolic cardiac function, enhanced myocardial CB2R expression, oxidative/nitrative stress, and pro-inflammatory response (tumor necrosis factor-α, interleukin-1ß, intracellular adhesion molecule 1, macrophage inflammatory protein-1, monocyte chemoattractant protein-1), macrophage infiltration, fibrosis, and cell death. Pharmacological activation of CB2R with a selective agonist attenuated diabetes-induced inflammation, oxidative/nitrative stress, fibrosis and cell demise, and consequent cardiac dysfunction without affecting hyperglycemia. In contrast, genetic deletion of CB2R aggravated myocardial pathology. Thus, selective activation of CB2R ameliorates diabetes-induced myocardial tissue injury and preserves the functional contractile capacity of the myocardium in the diabetic milieu. This is particularly encouraging, since unlike CB1R agonists, CB2R agonists do not elicit psychoactive activity and cardiovascular side effects and are potential clinical candidates in the treatment of diabetic cardiovascular and other complications.

Interplay of cardiovascular mediators, oxidative stress and inflammation in liver disease and its complications.

The liver is a crucial metabolic organ that has a key role in maintaining immune and endocrine homeostasis. Accumulating evidence suggests that chronic liver disease might promote the development of various cardiac disorders (such as arrhythmias and cardiomyopathy) and circulatory complications (including systemic, splanchnic and pulmonary complications), which can eventually culminate in clinical conditions ranging from portal and pulmonary hypertension to pulmonary, cardiac and renal failure, ascites and encephalopathy. Liver diseases can affect cardiovascular function during the early stages of disease progression. The development of cardiovascular diseases in patients with chronic liver failure is associated with increased morbidity and mortality, and cardiovascular complications can in turn affect liver function and liver disease progression. Furthermore, numerous infectious, inflammatory, metabolic and genetic diseases, as well as alcohol abuse can also influence both hepatic and cardiovascular outcomes. In this Review, we highlight how chronic liver diseases and associated cardiovascular effects can influence different organ pathologies. Furthermore, we explore the potential roles of inflammation, oxidative stress, vasoactive mediator imbalance, dysregulated endocannabinoid and autonomic nervous systems and endothelial dysfunction in mediating the complex interplay between the liver and the systemic vasculature that results in the development of the extrahepatic complications of chronic liver disease. The roles of ageing, sex, the gut microbiome and organ transplantation in this complex interplay are also discussed.

Long-Term PDE-5A Inhibition Improves Myofilament Function in Left and Right Ventricular Cardiomyocytes through Partially Different Mechanisms in Diabetic Rat Hearts.

Heart failure with preserved ejection fraction (HFpEF) and right ventricular (RV) dysfunction are frequent complications of diabetic cardiomyopathy. Here we aimed to characterize RV and left ventricular (LV) remodeling and its prevention by vardenafil (a long-acting phosphodiesterase-5A (PDE-5A) inhibitor) administration in a diabetic HFpEF model. Zucker Diabetic Fatty (ZDF) and control, ZDF Lean (Lean) male rats received 10 mg/kg vardenafil (ZDF + Vard; Lean + Vard) per os, on a daily basis for a period of 25 weeks. In vitro force measurements, biochemical and histochemical assays were employed to assess cardiomyocyte function and signaling. Vardenafil treatment increased cyclic guanosine monophosphate (cGMP) levels and decreased 3-nitrotyrosine (3-NT) levels in the left and right ventricles of ZDF animals, but not in Lean animals. Cardiomyocyte passive tension (Fpassive) was higher in LV and RV cardiomyocytes of ZDF rats than in those receiving preventive vardenafil treatment. Levels of overall titin phosphorylation did not differ in the four experimental groups. Maximal Ca2+-activated force (Fmax) of LV and RV cardiomyocytes were preserved in ZDF animals. Ca2+-sensitivity of isometric force production (pCa50) was significantly higher in LV (but not in RV) cardiomyocytes of ZDF rats than in their counterparts in the Lean or Lean + Vard groups. In accordance, the phosphorylation levels of cardiac troponin I (cTnI) and myosin binding protein-C (cMyBP-C) were lower in LV (but not in RV) cardiomyocytes of ZDF animals than in their counterparts of the Lean or Lean + Vard groups. Vardenafil treatment normalized pCa50 values in LV cardiomyocytes, and it decreased pCa50 below control levels in RV cardiomyocytes in the ZDF + Vard group. Our data illustrate partially overlapping myofilament protein alterations for LV and RV cardiomyocytes in diabetic rat hearts upon long-term PDE-5A inhibition. While uniform patterns in cGMP, 3-NT and Fpassive levels predict identical effects of vardenafil therapy for the diastolic function in both ventricles, the uneven cTnI, cMyBP-C phosphorylation levels and pCa50 values implicate different responses for the systolic function.