Increase In Fractional Shortening Research Articles

Discovery of Nelutroctiv (CK-136), a Selective Cardiac Troponin Activator for the Treatment of Cardiovascular Diseases Associated with Reduced Cardiac Contractility.

Cardiac myosin activation has been shown to be a viable approach for the treatment of heart failure with reduced ejection fraction. Here, we report the discovery of nelutroctiv (CK-136), a selective cardiac troponin activator intended for patients with cardiovascular conditions where cardiac contractility is reduced. Discovery of nelutroctiv began with a high-throughput screen that identified compound 1R, a muscle selective cardiac sarcomere activator devoid of phosphodiesterase-3 activity. Optimization of druglike properties for 1R led to the replacement of the sulfonamide and aniline substituents which resulted in improved pharmacokinetic (PK) profiles and a reduced potential for human drug-drug interactions. In vivo echocardiography assessment of the optimized leads showed concentration dependent increases in fractional shortening and an improved pharmacodynamic window compared to myosin activator CK-138. Overall, nelutroctiv was found to possess the desired selectivity, a favorable pharmacodynamic window relative to myosin activators, and a preclinical PK profile to support clinical development.

Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Injectable, stretchable, and conductance-stable fiber for myocardial infarction repair

Injecting electroactive biomaterials into infarcted myocardium to restore blocked electrical signal pathways is an effective way for repairing myocardial infarction. However, re-establishing stable and anisotropic conductive pathways during the constant contraction and relaxation of the heart remains a challenge. Herein, inspired by the myocardial fibers with striated surfaces, a super-elastic and conductance-stable polyurethane fiber with buckled polypyrrole coating (PU150 %@PPy) was designed. The bionic composite fiber exhibited excellent stretchability (>450 %), electrical stability under large strains (ΔR/R0 of only 0.04 at 100 % strain), and long-term durability (>1000000 cycles), enabling stable transmission of electrical signals in a beating heart. Specifically, the composite fibers could be injected into the infarct area in minimally invasive manner and aligned with the myocardial fibers to provide anisotropic conductive pathways. After injecting the PU@PPy fibers into the infarcted heart, the cardiac function was improved (33.19 % increase in fractional shortening and 38.80 % increase in ejection fraction) and the myocardial remodeling was decreased (40.30 % reduction in infarct size and 95.12 % increase in myocardial wall thickness) by re-establishing the electrical communication of the electrically separated cardiomyocytes in the infarct area as well as by providing mechanical support. The injectable, stretchable, and conductance-stable fiber offers a promising strategy for reconstructing infarcted myocardium.

Abstract P1079: Regeneration Of Primate Hearts With Human IPS Cell-derived Cardiac Spheroids

Background: Clinical application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for cardiac repair started with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of hiPSC-CMs into the cardiac muscle layer, which was shown to induce electrical integration, was unclear because of concerns regarding poor engraftment of dispersed cardiomyocytes (CMs) and post-transplant arrhythmias. Thus, in this study, we prepared clinical-grade purified hiPSC-CM aggregates, known as cardiac spheroids (CSs), and confirmed whether the direct injection of CSs could regenerate infarcted non-human primate hearts. Methods: We performed two separate experiments to explore the appropriate number of CMs derived from human induced pluripotent stem cells (hiPSCs). In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks pre-transplantation and were designated as recipients of CSs containing 2х10 7 CMs or the vehicle. The animals were killed 12 weeks post-transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6х10 7 CMs). Results: Recipients of CSs containing 2х10 7 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle, with a low incidence of post-transplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits. The incidence of post-transplant arrhythmia slightly increased in recipients of CSs containing 6х10 7 CMs. Conclusions: We demonstrated that direct injection of hiPSC-derived CSs, prepared as clinical-grade cell products, restores the contractile functions of injured primate hearts with an acceptable risk of post-transplant arrhythmia. These findings provide strong rationale for conducting clinical trials using the equivalent CM products.

Pit bull-type breeds with dilated cardiomyopathy eating nontraditional diets improve after diet change (2015-2022).

To compare signalment, clinical signs, diet, echocardiographic findings, and outcome for pit bull-type breeds diagnosed between 2015 and 2022 with dilated cardiomyopathy (DCM) or with DCM diagnosed by a cardiologist but that did not meet all study echocardiographic criteria (DCM-C). 91 dogs with DCM and 11 dogs with DCM-C. Data were collected on clinical findings, echocardiographic measurements, and diet at the time of diagnosis (for 76/91 dogs); echocardiographic changes; and survival. For dogs with diet information available for time of diagnosis, 64/76 (84%) dogs were eating nontraditional commercial diets, while 12/76 (16%) were eating traditional commercial diets. There were few differences between diet groups at baseline, with congestive heart failure and arrhythmias common in both groups. Thirty-four dogs with known baseline diet and diet change status had follow-up echocardiograms between 60 and 1,076 days later (traditional diet, n = 7; nontraditional diet that changed diets, 27; and nontraditional diet group without diet change, 0). Dogs in the nontraditional diet group that changed diets had a significantly greater decrease in normalized left ventricular diameter (diastolic, P = .02; systolic, P = .048) and the left atrium-to-aorta ratio (P = .002) and a significantly greater increase in fractional shortening (P = .02) compared to dogs eating traditional diets. Dogs eating nontraditional diets with diet change (n = 45; P < .001) and dogs eating traditional diets (12; P < .001) had a significantly longer survival time compared to dogs eating nontraditional diets without diet change (4). Dogs with DCM-C also had significant echocardiographic improvements after diet change. Congestive heart failure and arrhythmias were common in pit bull-type breeds with DCM. Those eating nontraditional diets that changed diets had significant improvements in echocardiographic measurements after diet change.

Injectable and conductive cardiac patches repair infarcted myocardium in rats and minipigs.

Cardiac patches can help to restore the electrophysiological properties of the heart after myocardial infarction. However, scaffolds for the repair of heart muscle typically require surgical implantation or, if they are injectable, they are not electrically conductive or do not maintain their shape or function. Here, we report the performance, as demonstrated for the repair of infarcted heart muscle in rats and minipigs, of injectable and conductive scaffolds consisting of methacrylated elastin and gelatin, and carbon nanotubes that display shape-memory behaviour, a hierarchical porous structure and a negligible Poisson's ratio. In rats, the implantation of cell-free patches or patches seeded with rat cardiomyocytes onto the myocardium after ligation of the left anterior descending coronary artery led to functional repair after 4 weeks, as indicated by increases in fractional shortening and the ejection fraction, and by a decrease in the infarcted area. We also observed measures of functional recovery in minipigs with infarcted hearts after the delivery of cell-free patches or patches incorporating cardiomyocytes differentiated from human pluripotent stem cells.

Dipeptidyl peptidase 4 inhibitors attenuate cardiac ischaemia-reperfusion injury in rats with diabetes mellitus type 2.

The aim of our study was to assess and compare the effects of dipeptidyl peptidase 4 (DPP4) inhibitors, saxagliptin and sitagliptin, on metabolic control of disease and cardiac function in rats with diabetes mellitus type 2 (T2DM). This research would provide novel understanding into the potentially protective effects of DPP4 inhibitors in helping salvage of the heart exposed to ischaemia-reperfusion (I-R) injury. Forty-eight Wistar albino rats were randomly divided into four groups: CTRL, Control healthy group; T2DM, rats with T2DM; T2DM+Sit, rats with T2DM treated with 0.6mg/kg of sitagliptin; T2DM+Sax, rats with T2DM treated with 0.45mg/kg of saxagliptin for 3weeks. At the end of the protocol, in vivo cardiac function was assessed by echocardiography, while in the blood samples glucose and insulin were determined. Additionally, ex vivo heart function was estimated on a model of I-R injury using Langendorff apparatus. Immunohistochemical analysis was used to determine the degree of myocardial apoptosis and necrosis, while DPP4 staining was performed to assess the cardiac DPP4 expression. Data were analyzed using a one-way analysis of variance (ANOVA) and the post hoc Bonferroni test for multiple comparisons. Improved glycoregulation was noticed in rats that received DPP4 inhibitors compared to untreated diabetic rats (P<.05). Moreover, better in vivo systolic function was observed in rats treated with both DPP4 inhibitors as evidenced by an increase in fractional shortening when compared to T2DM (P<.05). Most parameters of cardiac function in treated rats remained unaltered during reperfusion, thus suggesting that both drugs protected myocardium during flow restoration. Better effects on coronary circulation were achieved after sitagliptin application. Additionally, both DPP4 inhibitors showed similar potential to attenuate cardiac necrosis and apoptosis. Saxagliptin and sitagliptin might be efficient in preserving myocardial function and morphology in ex vivo induced I-R cardiac injury in rats with T2DM.

A Coupled Electromechanical Myocyte Model to Assess the Effects of 2‐deoxy‐ATP on Contractile Function in the Heart

IntroductionHeart failure (HF) remains a significant cause of morbidity, mortality, and medical costs. HF is characterized by reduced contractile function and inability of the heart to pump sufficient blood to meet the body’s demands. Existing treatments for HF do not permanently restore cardiac function and can lead to side effects such as impaired ventricular relaxation. 2‐deoxy‐ATP (dATP), a novel heart failure therapeutic, has been shown to increase contractile function while enhancing relaxation [1]. Previous studies have shown that dATP increases the rate of crossbridge cycling [2]. It has also been shown that dATP acts on the sarcoplasmic reticulum ATPase (SERCA), the pump responsible for removing calcium (Ca2+) from the cytosol, which could explain the enhanced relaxation kinetics seen experimentally [3]. However, the mechanism of dATP is still not fully understood. Further, it is not clear whether the increased rate of crossbridge cycling with dATP treatment could aggravate energy starvation in HF.MethodsTo assess the full mechanism of dATP at the cellular level, we utilized a myocyte model containing several ion channels, including SERCA [4]. We coupled this model to a mitochondria model to investigate whether dATP treatment aggravates energy starvation by tracking ATP, ADP, Pi, and oxygen concentrations over time [5]. We also coupled the myocyte model to a previously developed 5 state Markov model of crossbridge cycling, which captures regulation of contraction via Ca2+ binding to troponin [2]. The crossbridge cycling model was modified to capture the effects of sarcomere length on Ca2+ sensitivity. dATP treatment was simulated by adjusting the ATP and Ca2+ binding rates in the SERCA model and the rate of crossbridge cycling in the sarcomere model based on previous studies [2, 3].Results and ConclusionsWe found that the combined effects of dATP on both SERCA and the sarcomere within the coupled myocyte model could explain the increase in fractional shortening and time to 50% and 90% relaxation of the Ca2+ transient seen experimentally more accurately than either model alone. Additionally, we found no evidence that dATP aggravates energy starvation, affirming dATP as a promising therapeutic for HF.Support or Funding InformationNIH HLT32 TG

087 Blockade of VEGF-B Improves Cardiac Function after Myocardial Infarction in Insulin-Resistant Mice

Anti-vascular endothelial growth factor B (VEGF-B) strategies have recently been shown to reduce toxic lipid accumulation in heart and to improve cardiac glucose uptake in rodents. We now investigate whether anti-VEGF-B treatment improves heart function following acute myocardial ischaemia-reperfusion in the setting of type 2 diabetes where cardiac insulin responsiveness and glucose uptake are impaired. Insulin-resistant (High fat diet, HFD) mice were treated with anti-VEGF-B or control antibody (20 mg/kg, ip) twice weekly for 5 weeks including three weeks before and two weeks after surgically-induced myocardial infarction (MI). One hour post-reperfusion, cardiac glucose uptake was assessed by FDG-PET/CT and two weeks later left ventricular (LV) function was measured by echocardiography Two weeks post-myocardial infarction (MI) and in comparison with control, anti-VEGF-B treatment reduced infarct size by 29±6% (mean±SEM) and enhanced left ventricular systolic function as measured by a 19±4% increase in ejection fraction and a 38±6% increase in fractional shortening (p<0.05 for all). Anti-VEGF-B treatment also trended to limit post-ischemic left ventricular dilatation by reducing both end diastolic and end systolic volumes. This was aligned with the finding of lower heart mass after myocardial infarction after treatment with anti-VEGF-B antibody compared to control (p<0.05). However, anti-VEGF-B treatment did not significantly modulate cardiac glucose uptake of HFD mice one hour after myocardial infarction This study demonstrates that anti-VEGF-B therapy improves the left ventricular systolic function of insulin-resistant mice after myocardial infarction. The mechanisms underlying this effect remain to be determined.

Hemodynamic Stimulation Using the Biomimetic Cardiac Tissue Model (BCTM) Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell (hiPSC)-derived cardio­myocytes (hiPSC-CMs) hold great promise for cardiovascular disease modeling and regenerative medicine. However, these cells are both structurally and functionally ­immature, primarily due to their differentiation into cardiomyocytes occurring under static culture which only reproduces biomolecular cues and ignores the dynamic hemo­dynamic cues that shape early and late heart development during cardiogenesis. To evaluate the effects of hemodynamic stimuli on hiPSC-CM maturation, we used the biomimetic cardiac tissue model to reproduce the hemodynamics and pressure/volume changes associated with heart development. Following 7 days of gradually increasing stimulation, we show that hemodynamic loading results in (a) enhanced alignment of the cells and extracellular matrix, (b) significant increases in genes associated with physiological hypertrophy, (c) noticeable changes in sarcomeric organization and potential changes to cellular metabolism, and (d) a significant increase in fractional shortening, suggestive of a positive force frequency response. These findings suggest that culture of hiPSC-CMs under conditions that accurately reproduce hemodynamic cues results in structural orga­nization and molecular signaling consistent with organ growth and functional maturation.

Effects of hypoestrogenism and/or hyperaldosteronism on myocardial remodeling in female mice.

We investigated the potential adverse effects of hyperaldosteronism and/or hypoestrogenism on cardiac phenotype, and examined their combined effects in female mice overexpressing cardiac aldosterone synthase (AS). We focused on some signaling cascades challenging defensive responses to adapt and/or to survive in the face of double deleterious stresses, such as Ca2+‐homeostasis, pro/anti‐hypertrophic, endoplasmic reticulum stress (ER stress), pro‐ or anti‐apoptotic effectors, and MAP kinase activation, and redox signaling. These protein expressions were assessed by immunoblotting at 9 weeks after surgery. Female wild type (FWT) and FAS mice were fed with phytoestrogen‐free diet; underwent ovariectomy (Ovx) or sham‐operation (Sham). Ovx increased gain weight and hypertrophy index. Transthoracic echocardiograghy was performed. Both Ovx‐induced heart rate decrease and fractional shortening increase were associated with collagen type III shift. Cardiac estrogen receptor (ER α, ER β) protein expression levels were downregulated in Ovx mice. Hypoestrogenism increased plasma aldosterone and MR protein expression in FAS mice. Both aldosterone and Ovx played as mirror effects on up and downstream signaling effectors of calcium/redox homeostasis, apoptosis, such as concomitant CaMKII activation and calcineurin down–regulation, MAP kinase inhibition (ERK1/2, p38 MAPK) and Akt activation. The ratio Bcl2/Bax is in favor to promote cell survivor. Finally, myocardium had dynamically orchestrated multiple signaling cascades to restore tolerance to hostile environment thereby contributing to a better maintenance of Ca2+/redox homeostasis. Ovx‐induced collagen type III isoform shift and its upregulation may be important for the biomechanical transduction of the heart and the recovery of cardiac function in FAS mice. OVX antagonized aldosterone signaling pathways.

Subcutaneous Ehrlich Ascites Carcinoma mice model for studying cancer-induced cardiomyopathy

Cardiomyopathy is one of the characteristic features of cancer. In this study, we establish a suitable model to study breast cancer-induced cardiomyopathy in mice. We used Ehrlich Ascites Carcinoma cells to induce subcutaneous tumor in 129/SvJ mice and studied its effect on heart function. In Ehrlich Ascites Carcinoma bearing mice, we found significant reduction in left ventricle wall thickness, ejection fraction, and fractional shortening increase in left ventricle internal diameter. We found higher muscle atrophy, degeneration, fibrosis, expression of cell-adhesion molecules and cell death in tumor-bearing mice hearts. As observed in cancer patients, we found that mTOR, a key signalling molecule responsible for maintaining cell growth and autophagy was suppressed in this model. Tumor bearing mice hearts show increased expression and nuclear localization of TFEB and FoxO3a transcription factors, which are involved in the upregulation of muscle atrophy genes, lysosomal biogenesis genes and autophagy genes. We propose that Ehrlich Ascites Carcinoma induced tumor can be used as a model to identify potential therapeutic targets for the treatment of heart failure in patients suffering from cancer-induced cardiomyopathy. This model can also be used to test the adverse consequences of cancer chemotherapy in heart.

Hemodynamic Changes During Rewarming Phase of Whole-Body Hypothermia Therapy in Neonates with Hypoxic-Ischemic Encephalopathy

To delineate the systemic and cerebral hemodynamic response to incremental increases in core temperature during the rewarming phase of therapeutic hypothermia in neonatal hypoxic-ischemic encephalopathy (HIE). Continuous hemodynamic data, including heart rate (HR), mean arterial blood pressure (MBP), cardiac output by electrical velocimetry (COEV), arterial oxygen saturation, and renal (RrSO2) and cerebral (CrSO2) regional tissue oxygen saturation, were collected from 4 hours before the start of rewarming to 1 hour after the completion of rewarming. Serial echocardiography and transcranial Doppler were performed at 3 hours and 1 hour before the start of rewarming (T-3 and T-1; "baseline") and at 2, 4, and 7 hours after the start of rewarming (T+2, T+4, and T+7; "rewarming") to determine Cardiac output by echocardiography (COecho), stroke volume, fractional shortening, and middle cerebral artery (MCA) flow velocity indices. Repeated-measures analysis of variance was used for statistical analysis. Twenty infants with HIE were enrolled (mean gestational age, 38.8 ± 2 weeks; mean birth weight, 3346 ± 695 g). During rewarming, HR, COecho, and COEV increased from baseline to T+7, and MBP decreased. Despite an increase in fractional shortening, stroke volume remained unchanged. RrSO2 increased, and renal fractional oxygen extraction (FOE) decreased. MCA peak systolic flow velocity increased. There were no changes in CrSO2 or cerebral FOE. In neonates with HIE, CO significantly increases throughout rewarming. This is due to an increase in HR rather than stroke volume and is associated with an increase in renal blood flow. The lack of change in cerebral tissue oxygen saturation and extraction, in conjunction with an increase in MCA peak systolic velocity, suggests that cerebral flow metabolism coupling remained intact during rewarming.

In vivo efficacy of the AKT inhibitor ARQ 092 in Noonan Syndrome with multiple lentigines-associated hypertrophic cardiomyopathy.

Noonan Syndrome with Multiple Lentigines (NSML, formerly LEOPARD syndrome) is an autosomal dominant "RASopathy" disorder manifesting in congenital heart disease. Most cases of NSML are caused by catalytically inactivating mutations in the protein tyrosine phosphatase (PTP), non-receptor type 11 (PTPN11), encoding the SH2 domain-containing PTP-2 (SHP2) protein. We previously generated knock-in mice harboring the PTPN11 mutation Y279C, one of the most common NSML alleles; these now-termed SHP2Y279C/+ mice recapitulate the human disorder and develop hypertrophic cardiomyopathy (HCM) by 12 weeks of age. Functionally, heart and/or cardiomyocyte lysates from SHP2Y279C/+ mice exhibit increased basal and agonist-induced AKT and mTOR activities. Here, we sought to determine whether we could reverse the hypertrophy in SHP2Y279C/+ mice using ARQ 092, an oral and selective allosteric AKT inhibitor currently in clinical trials for patients with PI3K/AKT-driven tumors or Proteus syndrome. We obtained echocardiographs of SHP2Y279C/+ and wildtype (SHP2+/+) littermates, either in the presence or absence of ARQ 092 at 12, 14, and 16 weeks of age. While SHP2Y279C/+ mice developed significant left ventricular hypertrophy by 12 weeks, as indicated by decreased chamber dimension and increased posterior wall thickness, treatment of SHP2Y279C/+ mice with ARQ 092 normalized the hypertrophy in as early as 2 weeks following treatment, with hearts comparable in size to those in wildtype (SHP2+/+) mice. In addition, we observed an increase in fractional shortening (FS%) in SHP2Y279C/+ mice, an effect of increased compensatory hypertrophy, which was not apparent in SHP2Y279C/+ mice treated with ARQ 092, suggesting functional improvement of HCM upon treatment with the AKT inhibitor. Finally, we found that ARQ 092 specifically inhibited AKT activity, as well as its downstream effectors, PRAS and S6RP in NSML mice. Taken together, these data suggest ARQ 092 may be a promising novel therapy for treatment of hypertrophy in NSML patients.

Antioxidant Treatment Improves Cardiac Dysfunction in a Murine Model of Premature Aging.

Bmal1-(brain and muscle ARNT-like protein-1) deficient (Bmal1) mice prematurely age because of an increased reactive oxygen species (ROS) production. These mice also show a decline in cardiac function with age. We investigated whether an antioxidant treatment can ameliorate the declining cardiac function in prematurely aged Bmal1 mice. Male Bmal1 and wild-type (Bmal1) mice were exposed for 15 weeks to a high fat and high cholesterol diet with or without the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL; 5 mmol/L; in drinking water during the last 10 weeks). Echocardiographic analysis revealed that TEMPOL treatment of Bmal1 mice normalized cardiac function, as evidenced by a decrease in left ventricular diastolic and systolic internal diameters, and by an increase in fractional shortening and ejection fraction. The antioxidant did not affect cardiac function in Bmal1 mice. Although TEMPOL did not influence cardiac ROS levels in Bmal1 mice, it significantly protected Bmal1 cardiac telomeres from oxidation, as evidenced by a reduction in the telomere damage score (0.11 ± 0.012% vs. 0.16 ± 0.015%; P = 0.028). Thus, antioxidant treatment normalized cardiac function of Bmal1 mice, probably in part by scavenging ROS.

Anti-Atherogenic Properties of Allium ursinum Liophylisate: Impact on Lipoprotein Homeostasis and Cardiac Biomarkers in Hypercholesterolemic Rabbits.

The present investigation evaluates the capacity of Allium ursinum (wild garlic) leaf lyophilisate (WGLL; alliin content: 0.261%) to mitigate cardiovascular damage in hypercholesterolemic rabbits. New Zealand rabbits were divided into three groups: (i) cholesterol-free rabbit chow (control); (ii) rabbit chow containing 2% cholesterol (hypercholesterolemic, HC); (iii) rabbit chow containing 2% cholesterol + 2% WGLL (hypercholesterolemic treated, HCT); for eight weeks. At the zero- and eight-week time points, echocardiographic measurements were made, along with the determination of basic serum parameters. Following the treatment period, after ischemia-reperfusion injury, hemodynamic parameters were measured using an isolated working heart model. Western blot analyses of heart tissue followed for evaluating protein expression and histochemical study for the atheroma status determination. WGLL treatment mediated increases in fractional shortening; right ventricular function; peak systolic velocity; tricuspidal annular systolic velocity in live animals; along with improved aortic and coronary flow. Western blot analysis revealed WGLL-associated increases in HO-1 protein and decreases in SOD-1 protein production. WGLL-associated decreases were observed in aortic atherosclerotic plaque coverage, plasma ApoB and the activity of LDH and CK (creatine kinase) in plasma. Plasma LDL was also significantly reduced. The results clearly demonstrate that WGLL has complex cardioprotective effects, suggesting future strategies for its use in prevention and therapy for atherosclerotic disorders.

Metoprolol Reverses β-Adrenergic Remodeling in the Failing Right Ventricle of Pulmonary Artery Hypertensive (PAH) Rats

Heart failure (HF) is the leading cause of death in PAH patients, but under current guidelines there is no treatment directed specifically at the failing right ventricle (RV). In a model of RV HF induced by PAH we examined whether a selective β1AR blocker (BB) metoprolol could slow adverse myocardial remodeling.A single injection of monocrotaline (60 mg/kg, MCT) in male Wistar rats was used to induce PAH leading to RV HF. Control (CON) animals were injected with saline. MCT animals were given metoprolol (10 mg/kg/day, BB group) or placebo (MCT group) daily by oral administration from 15 days post injection. Langendorff-perfused hearts were used for RV myocyte isolation (for functional experiments) or preparation of RV myocardial homogenates (for protein expression via Western blotting). Experiments were performed on the day MCT animals showed HF signs or on the median MCT HF day for BB/CON groups.In response to selective β1AR stimulation (isoproterenol 100nM with 100nM ICI118,551) MCT RV myocytes had a blunted increase in fractional shortening and rate of relaxation compared with CON (P<0.05; n=5-20).This difference was absent in the BB myocytes. Similar trends were observed for Ca2+ transient amplitude and decay. Consistent with observations in LV failure, in MCT RV expression of β1AR and adenylyl cyclase V/VI was reduced, whereas that of Gαi3 and GRK2 was increased compared with CON (P<0.01). In all cases, metoprolol treatment normalizes protein expression. Interestingly, the expression of another protein which regulates the βAR pathway (caveolin 3) was reduced in MCT vs. CON (P<0.05) and normalized in BB. These results add support to the growing evidence for use of selective β1-blockers to reverse detrimental remodeling of the failing RV in PAH patients.

Comparative effects of urocortins and stresscopin on cardiac myocyte contractility

RationaleThere is a current need for the development of new therapies for patients with heart failure. ObjectiveWe test the effects of members of the corticotropin-releasing factor (CRF) family of peptides on myocyte contractility to validate them as potential heart failure therapeutics. Methods and resultsAdult feline left ventricular myocytes (AFMs) were isolated and contractility was assessed in the presence and absence of CRF peptides Urocortin 2 (UCN2), Urocortin 3 (UCN3), Stresscopin (SCP), and the β-adrenergic agonist isoproterenol (Iso). An increase in fractional shortening and peak Ca2+ transient amplitude was seen in the presence of all CRF peptides. A decrease in Ca2+ decay rate (Tau) was also observed at all concentrations tested. cAMP generation was measured by ELISA in isolated AFMs in response to the CRF peptides and Iso and significant production was seen at all concentrations and time points tested. ConclusionsThe CRF family of peptides effectively increases cardiac contractility and should be evaluated as potential novel therapeutics for heart failure patients.

Abstract 188: Contractile Function and Myofilament Proteins of the Naked Mole-rat Heart Show Resistance to Oxidative Stress

The naked mole-rat (NMR) is the longest-lived rodent, with a maximum lifespan of &gt;31 years. Unlike every other mammal studied to date, this species withstands cardiovascular structural and functional changes for at least 75% of its lifespan. Due to the intersection of oxidative stress, aging, and cardiovascular disease, we questioned if NMRs were more resistant to oxidative stress-induced cardiac dysfunction compared to short-lived mice. Echocardiography showed that 7 days after a 20 mg/kg dose of doxorubicin (DOX), mice had a 25% decline in fractional shortening (36 ± 1% to 27 ± 2%). In contrast, the fractional shortening of NMRs was unchanged with DOX treatment (27 ± 1%). Previously we observed that while basal cardiac function is low, NMRs have a robust cardiac reserve, displaying a 1.7 fold increase in fractional shortening under exercise-like conditions. DOX-treated NMRs had a significant reduction in their dobutamine response, signifying a diminished cardiac reserve. Intriguingly, we found no changes in phosphorylation or expression of myofilament proteins in the NMR heart with DOX treatment. Mice on the other hand, increased the phosphorylation of cardiac myosin binding protein-C and switched expression from predominantly α-myosin heavy chain to the β-isoform. Electron microscopy showed that DOX caused marked mitochondrial swelling and loss of cristae as well as massive cardiac myofibrillar disarray in mice. Conversely, DOX-treated NMRs had only slight alterations to myofilament structure. NMRs additionally had twofold higher levels of glutathione in their hearts, indicating a high antioxidant capacity. These findings reveal that long-lived NMRs are less susceptible to oxidative stress-induced cardiac dysfunction than mice. The NMR’s low basal cardiac function, unique regulation of myofilament proteins, and high glutathione levels may all be integral in the species’ ability to withstand oxidative damage and preserve cardiac function well into its third decade of life.

Cell-based delivery of dATP via gap junctions enhances cardiac contractility

The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is a promising strategy to treat myocardial infarction and reverse heart failure, but to date the contractile benefit in most studies remains modest. We have previously shown that the nucleotide 2-deoxyadenosine triphosphate (dATP) can substitute for ATP as the energy substrate for cardiac myosin, and increasing cellular dATP content by globally overexpressing ribonucleotide reductase (R1R2) can dramatically enhance cardiac contractility. Because dATP is a small molecule, we hypothesized that it would diffuse readily between cells via gap junctions and enhance the contractility of neighboring coupled wild type cells. To test this hypothesis, we performed studies with the goals of (1) validating gap junction-mediated dATP transfer in vitro and (2) investigating the use of R1R2-overexpressing hPSC-CMs in vivo as a novel strategy to increase cardiac function. We first performed intracellular dye transfer studies using dATP conjugated to fluorescein and demonstrated rapid gap junction-mediated transfer between cardiomyocytes. We then cocultured wild type cardiomyocytes with either cardiomyocytes or fibroblasts overexpressing R1R2 and saw more than a twofold increase in the extent and rate of contraction of wild type cardiomyocytes. Finally, we transplanted hPSC-CMs overexpressing R1R2 into healthy uninjured rat hearts and noted an increase in fractional shortening from 41±4% to 53±5% just five days after cell transplantation. These findings demonstrate that dATP is an inotropic factor that spreads between cells via gap junctions. Our data suggest that transplantation of dATP-producing hPSC-CMs could significantly increase the effectiveness of cardiac cell therapy.