A cross-species validation of single-beat metrics of cardiac contractility.

The assessment of left ventricular (LV) contractility in animal models is useful in various experimental paradigms, yet obtaining such measures is inherently challenging and surgically invasive. In a cross-species study using small and large animals, we comprehensively tested the agreement and validity of multiple single-beat surrogate metrics of LV contractility against the field-standard metrics derived from inferior vena cava occlusion (IVCO). Fifty-six rats, 27 minipigs and 11 conscious dogs underwent LV and arterial catheterization and were assessed for a range of single-beat metrics of LV contractility. All single-beat metrics were tested for the various underlying assumptions required to be considered a valid metric of cardiac contractility, including load-independency, sensitivity to inotropic stimulation, and ability to diagnose contractile dysfunction in cardiac disease. Of all examined single-beat metrics, only LV maximal pressure normalized to end-diastolic volume (EDV), end-systolic pressure normalized to EDV, and the maximal rate of rise of the LV pressure normalized to EDV showed a moderate-to-excellent agreement with their IVCO-derived reference measure and met all the underlying assumptions required to be considered as a valid cardiac contractile metric in both rodents and large-animal models. Our findings demonstrate that single-beat metrics can be used as a valid, reliable method to quantify cardiac contractile function in basic/preclinical experiments utilizing small- and large-animal models KEY POINTS: Validating and comparing indices of cardiac contractility that avoid caval occlusion would offer considerable advantages for the field of cardiovascular physiology. We comprehensively test the underlying assumptions of multiple single-beat indices of cardiac contractility in rodents and translate these findings to pigs and conscious dogs. We show that when performing caval occlusion is unfeasible, single-beat metrics can be utilized to accurately quantify cardiac inotropic function in basic and preclinical research employing various small and large animal species. We report that maximal left-ventricular (LV)-pressure normalized to end-diastolic volume (EDV), LV end-systolic pressure normalized to EDV and the maximal rate of rise of the LV pressure waveform normalized to EDV are the best three single-beat metrics to measure cardiac inotropic function in both small- and large-animal models.

Influence of right ventricular pressure and volume overload on right and left ventricular diastolic function.

BACKGROUND: Ventricular interdependence may account for altered ventricular mechanics in congenital heart disease. The present study aimed to identify differences in load-dependent right ventricular (RV)-left ventricular (LV) interactions in porcine models of pulmonary stenosis (PS) and pulmonary insufficiency (PI) by invasive admittance-derived hemodynamics in conjunction with noninvasive cardiovascular magnetic resonance (CMR). METHODS: Seventeen pigs were used in the study (7 with PS, 7 with PI, and 3 controls). Progressive PS was created by tightening a Teflon tape around the pulmonary artery, and PI was created by excising 2 leaflets of the pulmonary valve. Admittance catheterization data were obtained for the RV and LV at 10 to 12 weeks after model creation, with the animal ventilated under temporary diaphragm paralysis. CMR was performed in all animals immediately prior to pressure-volume catheterization. RESULTS: In the PS group, RV contractility was increased, manifested by increased end-systolic elastance (mean difference, 1.29 mm Hg/mL; 95% confidence interval [CI], 0.57-2.00 mm Hg/mL). However, in the PI group, no significant changes were observed in RV systolic function despite significant changes in RV diastolic function. In the PS group, LV end-systolic volume was significantly lower compared with controls (mean difference, 25.1 mL; 95% CI, -40.5 to -90.7 mL), whereas in the PI group, the LV showed diastolic dysfunction, demonstrated by an elevated isovolumic relaxation constant and ventricular stiffness (mean difference, 0.03 mL-1; 95% CI, -0.02 to 0.09 mL-1). CONCLUSIONS: The LV exhibits systolic dysfunction and noncompliance with PI. PS is associated with preserved LV systolic function and evidence of some LV diastolic dysfunction. Interventricular interactions influence LV filling and likely account for differential effects of RV pressure and volume overload on LV function.

Adaptive Reductions in Left Ventricular Diastolic Compliance Protect the Heart From Stretch-Induced Stunning.

Swine subjected to 2 weeks of repetitive pressure overload (RPO) exhibited significant myocyte loss, but left ventricular (LV) systolic function was preserved, and chamber dilatation did not occur. Instead, myocardial remodeling characterized by myocyte hypertrophy and interstitial fibrosis led to a marked reduction in LV diastolic compliance, which protected the heart from stretch-induced myocyte injury and preserved LV ejection fraction without anatomic LV hypertrophy. These results support a novel paradigm that links cardiac adaptations to RPO with the pathogenesis of reduced LV diastolic compliance and may explain how LV stiffening can occur in the absence of sustained hypertension or anatomic hypertrophy.

Improving Surgical Methods for Studying Vascular Grafts in Animal Models.

While clinical vascular grafting uses an end-to-side surgical method, researchers primarily use end-to-end implant techniques in preclinical models. This may be due in part to the limitations of using small animal models in research. The work presented here provides support and evidence for the improvement of vascular graft implant techniques by demonstrating the successful implantation of experimental grafts into both large and small animal models. Specifically, models of aortoiliac baboon (Papio anubis) bypass and common carotid rabbit (Oryctolagus cuniculus) bypass were used to test vascular grafts for thrombosis and vascular healing after 1 month using an end-to-side anastomosis grafting procedure. Patency was evaluated with ultrasound or histological techniques, and neointimal growth was quantified with histology. In the development of this procedure for small animals, both an end-to-end/end-to-side and an end-to-side/end-to-side configuration were tested in rabbits. One hundred percent of rabbit implants (2/2) with an end-to-end/end-to-side configuration were patent at explant. However, with the end-to-side/end-to-side configuration, 66% (6/9) of rabbit implants and 93% (13/14) of baboon implants remained patent at 1 month, suggesting the importance of replicating the end-to-side method for testing vascular grafts for clinical use. This study describes feasible preclinical surgical procedures, which simulate clinical vascular bypass grafts even in small animals. Widespread implementation of these end-to-side surgical techniques in these or other animals should improve the quality of experimental, preclinical testing and ultimately increase the likelihood of translating new vascular graft technologies into clinical applications.

Comparative Hemodynamic Effects of Contemporary Percutaneous Mechanical Circulatory Support Devices in a Porcine Model of Acute Myocardial Infarction.

OBJECTIVES: The aim of this study was to directly compare the hemodynamic effects of 2 contemporary percutaneous mechanical circulatory support devices in a porcine model of acute myocardial infarction. BACKGROUND: Percutaneous support devices offer the ability to unload the ischemic left ventricle, but the comparative hemodynamic effects of contemporary platforms are unclear. METHODS: Yorkshire swine (mean weight 76 ± 2 kg; n = 7) were instrumented with a left ventricular (LV) pressure-volume (PV) catheter and subjected to a 2-h coronary occlusion. Hemodynamic parameters and PV-derived indexes of LV performance were assessed 30 min after reperfusion and during LV support with Impella CP (ICP) and TandemHeart devices (in randomized order) at comparable flow rates. RESULTS: Myocardial infarction produced a rightward shift of the PV loop and increased LV end-diastolic pressure (from 9 ± 2 mm Hg to 15 ± 2 mm Hg; p = 0.04). After reperfusion, both devices maintained aortic pressure, shifted the PV loop to the left, and decreased LV end-diastolic pressure (ICP vs. TandemHeart; 11 ± 1 mm Hg vs. 7 ± 4 mm Hg; p = 0.04). However, only TandemHeart elicited significant reductions in native LV stroke volume (from 75 ± 7 ml to 39 ± 7 ml; p < 0.01), dP/dtmax (from 988 ± 77 mm Hg/s to 626 ± 42 mm Hg/s; p < 0.01), stroke work (from 0.70 ± 0.03 J to 0.26 ± 0.05 J; p < 0.01), PV area (from 0.95 ± 0.11 J to 0.47 ± 0.10 J; p < 0.01), and pre-load-recruitable stroke work slope (from 41.7 ± 2.8 J/ml to 30.6 ± 3.9 J/ml; p = 0.05). CONCLUSIONS: At comparable device flow rates, TandemHeart decreased LV pre-load, native LV stroke volume, and myocardial contractility to a greater degree than ICP. Reductions in load-independent indexes of LV performance indicate favorable effects on myocardial oxygen balance and support further study of TandemHeart in clinical scenarios requiring mechanical support in the setting of acute myocardial ischemia.

In-vivo characterization of left-ventricle pressure-volume telemetry system in swine model.

We present in-vivo study related to the use of our implantable RF telemetry system for pressure-volume (PV) cardiac monitoring in a animal subject. We implant a commercial MEMS PV sensor into the subject's heart left-ventricle (LV), while the telemetry system is implanted outside of the heart and connected to the sensor with a 7-microwires tether. The RF telemetry system is suitable for commercial application in medium sized subjects, its total volume of 2.475cm(3) and a weight of 4.0g. Our designed system is 58 % smaller in volume, 44 % in weight and has a 55 % reduction in sampling power over the last reported research in PV telemetry. In-vivo data was captured in both an acute and a freely moving setting over a 24 hour period. We experimentally demonstrated viability of the methodology that includes the surgical procedure and real-time monitoring of the in-vivo data in a freely moving subject. Further improvements in catheter design will improve the data quality and safety of the subject. This real-time implantable technology allows for researchers to quantify cardiac pathologies by extracting real-time pressure-volume loops, wirelessly from within freely moving subjects.

Parkinson-susceptibility gene DJ-1/PARK7 protects the murine heart from oxidative damage in vivo.

Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the ability of an organism to eliminate these toxic intermediates. Although the Parkinson-susceptibility gene, Parkinson protein 7/DJ-1 (DJ-1), has been linked to the regulation of oxidative stress, the exact mechanism by which this occurs and its in vivo relevance have remained elusive. In the heart, oxidative stress is a major contributor to the development of heart failure (HF). Therefore, we hypothesized that DJ-1 inhibits the pathological consequences of ROS production in the heart, the organ with the highest oxidative burden. We report that DJ-1 is highly expressed in normal heart tissue but is markedly reduced in end-stage human HF. DJ-1-deficient mice subjected to oxidative stress by transaortic banding exhibited exaggerated cardiac hypertrophy and susceptibility to developing HF. This was accompanied by a Trp53 (p53)-dependent decrease in capillary density, an excessive oxidation of DNA, and increased cardiomyocyte apoptosis, key events in the development of HF. Impaired mitochondrial biogenesis and progressive respiratory chain deficiency were also evident in cardiomyocytes lacking DJ-1. Our results provide compelling in vivo evidence that DJ-1 is a unique and nonredundant antioxidant that functions independent of other antioxidative pathways in the cellular defense against ROS.

Anti-apoptotic function of the E2F transcription factor 4 (E2F4)/p130, a member of retinoblastoma gene family in cardiac myocytes.

The E2F4-p130 transcriptional repressor complex is a cell-cycle inhibitor in mitotic cells. However, the role of E2F4/p130 in differentiated cells is largely unknown. We investigated the role of E2F4/p130 in the regulation of apoptosis in postmitotic cardiomyocytes. Here we demonstrate that E2F4 can inhibit hypoxia-induced cell death in isolated ventricular cardiomyocytes. As analyzed by chromatin immunoprecipitation, the E2F4-p130-repressor directly blocks transcription of essential apoptosis-related genes, E2F1, Apaf-1, and p73α through recruitment of histone deacetylase 1 (HDAC1). In contrast, diminution of the E2F4-p130-HDAC1-repressor and recruitment of E2F1 and histone acetylase activity to these E2F-regulated promoters is required for the execution of cell death. Expression of kinase-dead HDAC1.H141A or HDAC-binding deficient p130ΔHDAC1 abolishes the antiapoptotic effect of E2F4. Moreover, histological examination of E2F4(-/-) hearts revealed a markedly enhanced degree of cardiomyocyte apoptosis. Taken together, our genetic and biochemical data delineate an essential negative function of E2F4 in cardiac myocyte apoptosis.

Post-myocardial infarct p27 fusion protein intravenous delivery averts adverse remodelling and improves heart function and survival in rodents.

AIMS: P27Kip1 (p27) blocks cell proliferation through the inhibition of cyclin-dependent kinase 2 (cdk-2). Despite robust expression in the heart, little is known about the regulation and function of p27 in this terminally differentiated tissue. Previously, we demonstrated that p27 exerts anti-apoptotic and growth-inhibitory effects through interaction with casein kinase 2 (ck2) in neonatal rat cardiomyocytes. Here, we test the hypothesis that delivery of a transactivator of transcription (TAT)-p27 fusion protein (TAT.p27) will improve cardiac function and survival in a rat model of myocardial infarction (MI). METHODS AND RESULTS: Fisher rats underwent permanent left anterior descending ligation-induced MI followed by iv injection of TAT.p27 or TAT.LacZ (20 mg/kg) on Days 1 and 7 post-MI. Delivery of TAT.p27 was evaluated by western blot (WB) and immunofluorescence microscopy. Heart function was assessed by echocardiography and pressure-volume catheter. Apoptosis, hypertrophy, and fibrosis were detected by histochemistry and morphometry. WB confirmed gradual reduction in endogenous cardiac p27 levels following MI, with immunohistochemistry demonstrating successful delivery of TAT.p27 to the heart. At 48 h post-MI, cardiac apoptosis was decreased in rats treated with TAT.p27 when compared with saline- and TAT.LacZ-treated controls. At 28 days post-MI, rats treated with TAT.p27 manifested less cardiomyocyte hypertrophy and fibrosis, less diminished cardiac function, and greater survival. Additionally, p27KO mice undergoing experimental MI suffered an early increase in apoptosis with a larger infarct size and markedly reduced survival when compared with wild-type (WT) controls. CONCLUSION: These gain- and loss-of-function studies reveal a critical role for p27 in cardiac remodelling post-MI.

Uterine cells are recruited to the infarcted heart and improve cardiac outcomes in female rats.

We evaluated the hypothesis that uterine cells home to the heart after injury and improve cardiac outcomes. Premenopausal women have fewer cardiovascular complications than age-matched men, but the mechanisms responsible for this protection have not been conclusively identified. Hysterectomy was performed in young female rats (leaving the ovaries intact), and 7 days later the left coronary artery was ligated to produce a myocardial infarction (MI). Cardiac function at 28 days post-MI was measured using echocardiography. Fractional shortening was best in non-hysterectomized (non-Hx) females and lower in both Hx females and males. Uteri were then removed from GFP rats and heterotopically transplanted into non-GFP recipients to investigate homing of uterine cells to the infarcted myocardium. Seven days later, the uterine transplant recipients underwent coronary ligation. GFP(+) cells were found in the recipient hearts 7 days after MI and persisted for 6 months. Confocal analysis showed that homed uterine cells were located around blood vessels, suggesting their involvement in neovascularization. We then evaluated uterine cell transplantation by intravenously injecting GFP(+) uterine cells into Hx females immediately after MI. These GFP(+) cells were found to home to the injured myocardium, stimulate angiogenesis, improve cardiac function, and increase survival. This study demonstrates that uterine cells can home to the injured myocardium, enhance tissue repair, and prevent cardiac dysfunction. Uterine cells may play a role in the prevention of cardiovascular complications in females.

PTEN-inducible kinase 1 (PINK1)/Park6 is indispensable for normal heart function.

Oxidative stress is caused by an imbalance between reactive oxygen species (ROS) production and the ability of an organism to eliminate these toxic intermediates. Mutations in PTEN-inducible kinase 1 (PINK1) link mitochondrial dysfunction, increased sensitivity to ROS, and apoptosis in Parkinson's disease. Whereas PINK1 has been linked to the regulation of oxidative stress, the exact mechanism by which this occurs has remained elusive. Oxidative stress with associated mitochondrial dysfunction leads to cardiac dysfunction and heart failure (HF). We hypothesized that loss of PINK1 in the heart would have deleterious consequences on mitochondrial function. Here, we observed that PINK1 protein levels are markedly reduced in end-stage human HF. We also report that PINK1 localizes exclusively to the mitochondria. PINK1(-/-) mice develop left ventricular dysfunction and evidence of pathological cardiac hypertrophy as early as 2 mo of age. Of note, PINK1(-/-) mice have greater levels of oxidative stress and impaired mitochondrial function. There were also higher degrees of fibrosis, cardiomyocyte apoptosis, and a reciprocal reduction in capillary density associated with this baseline cardiac phenotype. Collectively, our in vivo data demonstrate that PINK1 activity is crucial for postnatal myocardial development, through its role in maintaining mitochondrial function, and redox homeostasis in cardiomyocytes. In conclusion, PINK1 possesses a distinct, nonredundant function in the surveillance and maintenance of cardiac tissue homeostasis.

Infarct stabilization and cardiac repair with a VEGF-conjugated, injectable hydrogel.

Injectable scaffolds made of biodegradable biomaterials can stabilize a myocardial infarct and promote cardiac repair. Here, we describe the synthesis of a new, temperature-sensitive, aliphatic polyester hydrogel (HG) conjugated with vascular endothelial growth factor (VEGF) and evaluate its effects on cardiac recovery after a myocardial infarction (MI). Seven days after coronary ligation in rats, PBS, HG, or HG mixed or conjugated with VEGF (HG + VEGF or HG-VEGF, respectively) was injected around the infarct (n = 8-11/group). Function was evaluated by echocardiography at multiple time points. Pressure-volume measurements were taken and infarct morphometry and blood vessel density were assessed at 35 days after injection. HG-VEGF provided localized, sustained VEGF function. Compared with outcomes in the PBS group, fractional shortening, ventricular volumes, preload recruitable stroke work, and end-systolic elastance were all preserved (p < 0.05) in the HG and HG + VEGF groups, and further preserved in the HG-VEGF group. Conjugated VEGF also produced the highest blood vessel density (p < 0.05). The infarct thinned and dilated after PBS injection, but was smaller and thicker in hearts treated with HG (p < 0.05). Our temperature-sensitive HG attenuated adverse cardiac remodeling and improved ventricular function when injected after an MI. VEGF delivery enhanced these effects when the VEGF was conjugated to the HG.

Review of cellular and molecular pathways linking thrombosis and innate immune system during sepsis.

Cellular and molecular pathways link thrombosis and innate immune system during sepsis. Extrinsic pathway activation of protease thrombin through FVIIa and tissue factor (TF) in sepsis help activate its endothelial cell (EC) membrane Protease Activated Receptor 1 (PAR-1). Thrombin adjusts the EC cycle through activation of G proteins (G12/13), and later through Rho GEFs (guanine nucleotide exchange factors), and provides a path for Rho GTPases mediated cytoskeletal responses involved in shape change and permeability of the EC membrane leading to an increase of leakage of plasma proteins.At the same time, thrombin stimulates spontaneous mitogenesis by inducing activation of the cell cycle from G0-G1 to S by down-regulation of p27Kip1, a negative regulator of the cell cycle, in association with the up-regulation of S-phase kinase associated protein 2 (Skp2). After transport in cytoplasm, p27 Kip1 binds to RhoA thus prevent activation of RhoA by GEFs, thus inhibit GDP-GTP exchange mediated by GEFs. In cytoplasm, releasing factor (RF) p27-RF-Rho is able to free RhoA. P27 RF-Rho binds p27kip1 and prevents p27kip1 from binding to RhoA. Exposed RhoA is later able to increase the expression of the F-box protein Skp2, after its Akt triggered 14-3-3-ß-dependent cytoplasm relocation. Skp2 increases cytoplasm ubiquitination-dependent degradation of p27Kip1. Additionally, after septic induction of canonical NF-kB pathway in EC through TLR4/IRAK4/TRAF/IkB, an IKKα dimer phosphorylates the p52 precursor NF-kB2/p100, leading to p100 processing and translocation of RelB/p52 to the nucleus. By controlling the NF-kB-RelB complex, IKKα signaling regulates the transcription of the Skp2 and correspondingly p27Kip1.

Inherited trombophilic states and pulmonary embolism.

Pulmonary embolism (PE) and deep vein thrombosis (DVT) are associated with considerable morbidity and mortality, mostly, in case of PE for its lack of sensitivity of its early detection. For as much as twenty-five percent of PE patients the primary clinical appearance is unexpected death. While PE is one of the most avertable causes of hospital associated deaths, its diagnostics can be extremely difficult. Newly increased interest in an inherited thrombophilic states has been provoked by the discovery of several common inherited abnormalities, i.e. the prothrombin (PT) gene G20210A, Factor V Leiden (FVL) mutation (Arg506Gln), hyperhomocystenemia and homocysteiuria, Wein-Penzing defect, Sticky Platelet Syndrome (SPS), Quebec platelet disorder (QPD) and Sickle Cell Disease (SCD). PE incidence rates increase exponentially with age for both men and women, as they might harbor more than one thrombophilic state. Although the impact of genetic factors on PE is to some extent documented with lacking taxonomy, its genetic testing as its prevention strategy fall short.In this review thrombophilic states are divided into inherited or acquired, and only the inherited and newly documented are more closely followed. Factors are further grouped based on its thrombophilic taxonomy into; inherited defects of coagulation, inherited defects of fibrinolysis, inherited defects of enzymatic pathway in relation to development of VTE and PE and inherited defects of platelets in relation to PE. It was beyond the scope of this review to follow all inherited and newly recognized factors and its association to VTE and PE; however the overall taxonomy makes this review clinically valuable i.e. in relation to genetic testing as PE prevention.

Biodistribution of covalent antithrombin-heparin complexes.

We have developed a covalent antithrombin-heparin (ATH) complex with advantages compared to non-covalent antithrombin:heparin (AT:H) mixtures. In addition to increased activity, ATH has a longer intravenous half-life that is partly due to reduced plasma protein binding. Given ATH's altered clearance, we investigated biodistribution of ATH in vivo. ATH made from either human plasma-derived AT (pATH) or recombinant human (produced in goats) AT (rhATH) was studied. 125I-ATH + unlabeled carrier was injected into rabbits at different doses. 131I-labeled albumin was administered just before sacrifice as a marker for trapped blood in tissues. Immediately after sacrifice, animal components were removed, weighed, and subsamples were counted for gamma-radioactivity. Percent recoveries of ATH in various organs/compartments at different time points were calculated, and kinetic distribution plots generated. At saturating doses, early disappearance of rhATH from the circulation was much faster than pATH. Co-incident with clearance, 26 +/- 3% of dose for rhATH was liver-associated compared to only 3.7 +/- 0.5% for pATH by 20 min. Also, at early time periods, >60% of all extravascular ATH was liver-associated. Analysis of the vena cava and aorta suggested that vessel wall binding might also account for initial plasma loss of rhATH. By 24 h, most of pATH and rhATH were present as urinary degradation products (51 +/- 3% and 63 +/- 8%, respectively). In summary, systemic elimination of ATH is greatly influenced by the form of AT in the complex, with liver uptake and degradation playing a major role.