PKN2 deficiency leads both to prenatal 'congenital' cardiomyopathy and defective angiotensin II stress responses.

The protein kinase PKN2 is required for embryonic development and PKN2 knockout mice die as a result of failure in the expansion of mesoderm, cardiac development and neural tube closure. In the adult, cardiomyocyte PKN2 and PKN1 (in combination) are required for cardiac adaptation to pressure-overload. The specific role of PKN2 in contractile cardiomyocytes during development and its role in the adult heart remain to be fully established. We used mice with cardiomyocyte-directed knockout of PKN2 or global PKN2 haploinsufficiency to assess cardiac development and function using high resolution episcopic microscopy, MRI, micro-CT and echocardiography. Biochemical and histological changes were also assessed. Cardiomyocyte-directed PKN2 knockout embryos displayed striking abnormalities in the compact myocardium, with frequent myocardial clefts and diverticula, ventricular septal defects and abnormal heart shape. The sub-Mendelian homozygous knockout survivors developed cardiac failure. RNASeq data showed up-regulation of PKN2 in patients with dilated cardiomyopathy, suggesting an involvement in adult heart disease. Given the rarity of homozygous survivors with cardiomyocyte-specific deletion of PKN2, the requirement for PKN2 in adult mice was explored using the constitutive heterozygous PKN2 knockout. Cardiac hypertrophy resulting from hypertension induced by angiotensin II was reduced in these haploinsufficient PKN2 mice relative to wild-type littermates, with suppression of cardiomyocyte hypertrophy and cardiac fibrosis. It is concluded that cardiomyocyte PKN2 is essential for heart development and the formation of compact myocardium and is also required for cardiac hypertrophy in hypertension. Thus, PKN signalling may offer therapeutic options for managing congenital and adult heart diseases.

Phase 0 Study of Vandetanib-Eluting Radiopaque Embolics as a Preoperative Embolization Treatment in Patients with Resectable Liver Malignancies.

PURPOSE: To assess the safety and tolerability of a vandetanib-eluting radiopaque embolic (BTG-002814) for transarterial chemoembolization (TACE) in patients with resectable liver malignancies. MATERIALS AND METHODS: The VEROnA clinical trial was a first-in-human, phase 0, single-arm, window-of-opportunity study. Eligible patients were aged ≥18 years and had resectable hepatocellular carcinoma (HCC) (Child-Pugh A) or metastatic colorectal cancer (mCRC). Patients received 1 mL of BTG-002814 transarterially (containing 100 mg of vandetanib) 7-21 days prior to surgery. The primary objectives were to establish the safety and tolerability of BTG-002814 and determine the concentrations of vandetanib and the N-desmethyl vandetanib metabolite in the plasma and resected liver after treatment. Biomarker studies included circulating proangiogenic factors, perfusion computed tomography, and dynamic contrast-enhanced magnetic resonance imaging. RESULTS: Eight patients were enrolled: 2 with HCC and 6 with mCRC. There was 1 grade 3 adverse event (AE) before surgery and 18 after surgery; 6 AEs were deemed to be related to BTG-002814. Surgical resection was not delayed. Vandetanib was present in the plasma of all patients 12 days after treatment, with a mean maximum concentration of 24.3 ng/mL (standard deviation ± 13.94 ng/mL), and in resected liver tissue up to 32 days after treatment (441-404,000 ng/g). The median percentage of tumor necrosis was 92.5% (range, 5%-100%). There were no significant changes in perfusion imaging parameters after TACE. CONCLUSIONS: BTG-002814 has an acceptable safety profile in patients before surgery. The presence of vandetanib in the tumor specimens up to 32 days after treatment suggests sustained anticancer activity, while the low vandetanib levels in the plasma suggest minimal release into the systemic circulation. Further evaluation of this TACE combination is warranted in dose-finding and efficacy studies.

Options for imaging cellular therapeutics in vivo: a multi-stakeholder perspective.

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics.

Beetroot juice lowers blood pressure and improves endothelial function in pregnant eNOS-/- mice: importance of nitrate-independent effects.

KEY POINTS: Maternal hypertension is associated with increased rates of pregnancy pathologies, including fetal growth restriction, due at least in part to reductions in nitric oxide (NO) bioavailability and associated vascular dysfunction. Dietary nitrate supplementation, from beetroot juice (BRJ), has been shown to increase NO bioavailability and improve cardiovascular function in both preclinical and clinical studies. This study is the first to investigate effects of dietary nitrate supplementation in a pregnant animal model. Importantly, the effects of nitrate-containing BRJ were compared with both 'placebo' (nitrate-depleted) BRJ as well as water to control for potential nitrate-independent effects. Our data show novel, nitrate-independent effects of BRJ to lower blood pressure and improve vascular function in endothelial nitric oxide synthase knockout (eNOS-/- ) mice. These findings suggest potential beneficial effects of BRJ supplementation in pregnancy, and emphasize the importance of accounting for nitrate-independent effects of BRJ in study design and interpretation. ABSTRACT: Maternal hypertension is associated with adverse pregnancy outcomes, including fetal growth restriction (FGR), due in part to reductions in nitric oxide (NO) bioavailability. We hypothesized that maternal dietary nitrate administration would increase NO bioavailability to reduce systolic blood pressure (SBP), improve vascular function and increase fetal growth in pregnant endothelial NO synthase knockout (eNOS-/- ) mice, which exhibit hypertension, endothelial dysfunction and FGR. Pregnant wildtype (WT) and eNOS-/- mice were supplemented with nitrate-containing beetroot juice (BRJ+) from gestational day (GD) 12.5. Control mice received an equivalent dose of nitrate-depleted BRJ (BRJ-) or normal drinking water. At GD17.5, maternal SBP was measured; at GD18.5, maternal nitrate/nitrite concentrations, uterine artery (UtA) blood flow and endothelial function were assessed, and pregnancy outcomes were determined. Plasma nitrate concentrations were increased in both WT and eNOS-/- mice supplemented with BRJ+ (P < 0.001), whereas nitrite concentrations were increased only in eNOS-/- mice (P < 0.001). BRJ- did not alter nitrate/nitrite concentrations. SBP was lowered and UtA endothelial function was enhanced in eNOS-/- mice supplemented with either BRJ+ or BRJ-, indicating nitrate-independent effects of BRJ. Improvements in endothelial function in eNOS-/- mice were abrogated in the presence of 25 mm KCl, implicating enhanced EDH signalling in BRJ- treated animals. At GD18.5, eNOS-/- fetuses were significantly smaller than WT animals (P < 0.001), but BRJ supplementation did not affect fetal weight. BRJ may be a beneficial intervention in pregnancies associated with hypertension, endothelial dysfunction and reduced NO bioavailability. Our data showing biological effects of non-nitrate components of BRJ have implications for both interpretation of previous findings and in the design of future clinical trials.

Auxetic Cardiac Patches with Tunable Mechanical and Conductive Properties toward Treating Myocardial Infarction.

An auxetic conductive cardiac patch (AuxCP) for the treatment of myocardial infarction (MI) is introduced. The auxetic design gives the patch a negative Poisson's ratio, providing it with the ability to conform to the demanding mechanics of the heart. The conductivity allows the patch to interface with electroresponsive tissues such as the heart. Excimer laser microablation is used to micropattern a re-entrant honeycomb (bow-tie) design into a chitosan-polyaniline composite. It is shown that the bow-tie design can produce patches with a wide range in mechanical strength and anisotropy, which can be tuned to match native heart tissue. Further, the auxetic patches are conductive and cytocompatible with murine neonatal cardiomyocytes in vitro. Ex vivo studies demonstrate that the auxetic patches have no detrimental effect on the electrophysiology of both healthy and MI rat hearts and conform better to native heart movements than unpatterned patches of the same material. Finally, the AuxCP applied in a rat MI model results in no detrimental effect on cardiac function and negligible fibrotic response after two weeks in vivo. This approach represents a versatile and robust platform for cardiac biomaterial design and could therefore lead to a promising treatment for MI.

Direct comparison of high-temporal-resolution CINE MRI with Doppler ultrasound for assessment of diastolic dysfunction in mice.

Diastolic dysfunction is a sensitive early indicator of heart failure and can provide additional data to conventional measures of systolic function. Transmitral Doppler ultrasound, which measures the one-dimensional flow of blood through the mitral valve, is currently the preferred method for the measurement of diastolic function, but the measurement of the left ventricular volume changes using high-temporal-resolution cinematic magnetic resonance imaging (CINE MRI) is an alternative approach which is emerging as a potentially more robust and user-independent technique. Here, we investigated the performance of high-temporal-resolution CINE MRI and compared it with ultrasound for the detection of diastolic dysfunction in a mouse model of myocardial infarction. An in-house, high-temporal-resolution, retrospectively gated CINE sequence was developed with a temporal resolution of 1 ms. Diastolic function in mice was assessed using a custom-made, open-source reconstruction package. Early (E) and late (A) left ventricular filling phases were easily identifiable, and these measurements were compared directly with high-frequency, pulsed-wave, Doppler ultrasound measurements of mitral valve inflow. A repeatability study established that high-temporal-resolution CINE MRI and Doppler ultrasound showed comparable accuracy when measuring E/A in normal control mice. However, when applied in a mouse model of myocardial infarction, high-temporal-resolution CINE MRI indicated diastolic heart failure (E/A = 0.94 ± 0.11), whereas ultrasound falsely detected normal cardiac function (E/A = 1.21 ± 0.11). The addition of high-temporal-resolution CINE MRI to preclinical imaging studies enhances the library of sequences available to cardiac researchers and potentially identifies diastolic heart failure early in disease progression.

On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury.

The role of peroxisome proliferator-activated receptor α (PPARα)-mediated metabolic remodeling in cardiac adaptation to hypoxia has yet to be defined. Here, mice were housed in hypoxia for 3 wk before in vivo contractile function was measured using cine MRI. In isolated, perfused hearts, energetics were measured using (31)P magnetic resonance spectroscopy (MRS), and glycolysis and fatty acid oxidation were measured using [(3)H] labeling. Compared with a normoxic, chow-fed control mouse heart, hypoxia decreased PPARα expression, fatty acid oxidation, and mitochondrial uncoupling protein 3 (UCP3) levels, while increasing glycolysis, all of which served to maintain normal ATP concentrations ([ATP]) and thereby, ejection fractions. A high-fat diet increased cardiac PPARα expression, fatty acid oxidation, and UCP3 levels with decreased glycolysis. Hypoxia was unable to alter the high PPARα expression or reverse the metabolic changes caused by the high-fat diet, with the result that [ATP] and contractile function decreased significantly. The adaptive metabolic changes caused by hypoxia in control mouse hearts were found to have occurred already in PPARα-deficient (PPARα(-/-)) mouse hearts and sustained function in hypoxia despite an inability for further metabolic remodeling. We conclude that decreased cardiac PPARα expression is essential for adaptive metabolic remodeling in hypoxia, but is prevented by dietary fat.-Cole, M. A., Abd Jamil, A. H., Heather, L. C., Murray, A. J., Sutton, E. R., Slingo, M., Sebag-Montefiore, L., Tan, S. C., Aksentijevic, D., Gildea, O. S., Stuckey, D. J., Yeoh, K. K., Carr, C. A., Evans, R. D., Aasum, E., Schofield, C. J., Ratcliffe, P. J., Neubauer, S., Robbins, P. A., Clarke, K. On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury.

Use of high-frequency ultrasound to study the prenatal development of cranial neural tube defects and hydrocephalus in Gldc-deficient mice.

OBJECTIVE: We used non-invasive high-frequency ultrasound (HFUS) imaging to investigate embryonic brain development in a mouse model for neural tube defects (NTDs) and non-ketotic hyperglycinemia (NKH). METHOD: Using HFUS, we imaged embryos carrying loss of function alleles of Gldc encoding glycine decarboxylase, a component of the glycine cleavage system in mitochondrial folate metabolism, which is known to be associated with cranial NTDs and NKH in humans. We serially examined the same litter during the second half of embryonic development and quantified cerebral structures. Genotype was confirmed using PCR. Histology was used to confirm ultrasound findings. RESULTS: High-frequency ultrasound allowed in utero detection of two major brain abnormalities in Gldc-deficient mouse embryos, cranial NTDs (exencephaly) and ventriculomegaly (corresponding with the previous finding of post-natal hydrocephalus). Serial ultrasound allowed individual embryos to be analysed at successive gestational time points. From embryonic day 16.5 to 18.5, the lateral ventricle volume reduced in wild-type and heterozygous embryos but increased in homozygous Gldc-deficient embryos. CONCLUSION: Exencephaly and ventriculomegaly were detectable by HFUS in homozygous Gldc-deficient mouse embryos indicating this to be an effective tool to study CNS development. Longitudinal analysis of the same embryo allowed the prenatal onset and progression of ventricle enlargement in Gldc-deficient mice to be determined. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Origins of the vagal drive controlling left ventricular contractility.

KEY POINTS: The strength, functional significance and origins of parasympathetic innervation of the left ventricle remain controversial. This study tested the hypothesis that parasympathetic control of left ventricular contractility is provided by vagal preganglionic neurones of the dorsal motor nucleus (DVMN). Under ß-adrenoceptor blockade combined with spinal cord (C1) transection (to remove sympathetic influences), systemic administration of atropine increased left ventricular contractility in rats anaesthetized with urethane, confirming the existence of a tonic inhibitory muscarinic influence on cardiac inotropy. Increased left ventricular contractility in anaesthetized rats was observed when DVMN neurones were silenced. Functional neuroanatomical mapping revealed that vagal preganglionic neurones that have an impact on left ventricular contractility are located in the caudal region of the left DVMN. These neurones provide functionally significant parasympathetic control of left ventricular inotropy. ABSTRACT: The strength, functional significance and origins of direct parasympathetic innervation of the left ventricle (LV) remain controversial. In the present study we used an anaesthetized rat model to first confirm the presence of tonic inhibitory vagal influence on LV inotropy. Using genetic neuronal targeting and functional neuroanatomical mapping we tested the hypothesis that parasympathetic control of LV contractility is provided by vagal preganglionic neurones located in the dorsal motor nucleus (DVMN). It was found that under systemic ß-adrenoceptor blockade (atenolol) combined with spinal cord (C1) transection (to remove sympathetic influences), intravenous administration of atropine increases LV contractility in rats anaesthetized with urethane, but not in animals anaesthetized with pentobarbital. Increased LV contractility in rats anaesthetized with urethane was also observed when DVMN neurones targeted bilaterally to express an inhibitory Drosophila allatostatin receptor were silenced by application of an insect peptide allatostatin. Microinjections of glutamate and muscimol to activate or inhibit neuronal cell bodies in distinct locations along the rostro-caudal extent of the left and right DVMN revealed that vagal preganglionic neurones, which have an impact on LV contractility, are located in the caudal region of the left DVMN. Changes in LV contractility were only observed when this subpopulation of DVMN neurones was activated or inhibited. These data confirm the existence of a tonic inhibitory muscarinic influence on LV contractility. Activity of a subpopulation of DVMN neurones provides functionally significant parasympathetic control of LV contractile function.

Computational modeling of Takotsubo cardiomyopathy: effect of spatially varying ß-adrenergic stimulation in the rat left ventricle.

In Takotsubo cardiomyopathy, the left ventricle shows apical ballooning combined with basal hypercontractility. Both clinical observations in humans and recent experimental work on isolated rat ventricular myocytes suggest the dominant mechanisms of this syndrome are related to acute catecholamine overload. However, relating observed differences in single cells to the capacity of such alterations to result in the extreme changes in ventricular shape seen in Takotsubo syndrome is difficult. By using a computational model of the rat left ventricle, we investigate which mechanisms can give rise to the typical shape of the ventricle observed in this syndrome. Three potential dominant mechanisms related to effects of ß-adrenergic stimulation were considered: apical-basal variation of calcium transients due to differences in L-type and sarco(endo)plasmic reticulum Ca(2+)-ATPase activation, apical-basal variation of calcium sensitivity due to differences in troponin I phosphorylation, and apical-basal variation in maximal active tension due to, e.g., the negative inotropic effects of p38 MAPK. Furthermore, we investigated the interaction of these spatial variations in the presence of a failing Frank-Starling mechanism. We conclude that a large portion of the apex needs to be affected by severe changes in calcium regulation or contractile function to result in apical ballooning, and smooth linear variation from apex to base is unlikely to result in the typical ventricular shape observed in this syndrome. A failing Frank-Starling mechanism significantly increases apical ballooning at end systole and may be an important additional factor underpinning Takotsubo syndrome.

CT-Visible Microspheres Enable Whole-Body In Vivo Tracking of Injectable Tissue Engineering Scaffolds.

Targeted delivery and retention are essential requirements for implantable tissue-engineered products. Non-invasive imaging methods that can confirm location, retention, and biodistribution of transplanted cells attached to implanted tissue engineering scaffolds will be invaluable for the optimization and enhancement of regenerative therapies. To address this need, an injectable tissue engineering scaffold consisting of highly porous microspheres compatible with transplantation of cells is modified to contain the computed tomography (CT) contrast agent barium sulphate (BaSO4). The trackable microspheres show high x-ray absorption, with contrast permitting whole-body tracking. The microspheres are cellularized with GFP+ Luciferase+ mesenchymal stem cells and show in vitro biocompatibility. In vivo, cellularized BaSO4-loaded microspheres are delivered into the hindlimb of mice where they remain viable for 14 days. Co-registration of 3D-bioluminescent imaging and µCT reconstructions enable the assessment of scaffold material and cell co-localization. The trackable microspheres are also compatible with minimally-invasive delivery by ultrasound-guided transthoracic intramyocardial injections in rats. These findings suggest that BaSO4-loaded microspheres can be used as a novel tool for optimizing delivery techniques and tracking persistence and distribution of implanted scaffold materials. Additionally, the microspheres can be cellularized and have the potential to be developed into an injectable tissue-engineered combination product for cardiac regeneration.

Injectable biodegradable microcarriers for iPSC expansion and cardiomyocyte differentiation.

Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported. This is likely to be the result of low cell retention and survival following transplantation. This study aimed to improve the delivery and engraftment of viable cells by using an injectable microcarrier that provides an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). We describe the fabrication and characterisation of Thermally Induced Phase Separation (TIPS) microcarriers and their surface modification to enable iPSC-derived cardiomyocyte attachment in xeno-free conditions is described. The selected formulation resulted in iPSC attachment, expansion, and retention of pluripotent phenotype. Differentiation of iPSC into cardiomyocytes on the microcarriers is investigated in comparison with culture on 2D tissue culture plastic surfaces. Microcarrier culture is shown to support culture of a mature cardiomyocyte phenotype, be compatible with injectable delivery, and reduce anoikis. The findings from this study demonstrate that TIPS microcarriers provide a supporting matrix for culturing iPSC and iPSC-derived cardiomyocytes in vitro and are suitable as an injectable cell-substrate for cardiac regeneration.

Changes in cardiac-driven perivascular fluid movement around the MCA in a pharmacological model of acute hypertension detected with non-invasive MRI.

Perivascular spaces mediate a complex interaction between cerebrospinal fluid and brain tissue that may be an important pathway for solute waste clearance. Their structural or functional derangement may contribute to the development of age-related neurogenerative conditions. Here, we employed a non-invasive low b-value diffusion-weighted ECG-gated MRI method to capture perivascular fluid movement around the middle cerebral artery of the anaesthetised rat brain. Using this method, we show that such MRI estimates of perivascular fluid movement directionality are highly sensitive to the cardiac cycle. We then show that these measures of fluid movement directionality are decreased in the angiotensin-II pharmacological model of acute hypertension, with an associated dampening of vessel pulsatility. This translational MRI method may, therefore, be useful to monitor derangement of perivascular fluid movement associated with cardiovascular pathologies, such as hypertension, in order to further our understanding of perivascular function in neurology.

Layered rare-earth hydroxides as multi-modal medical imaging probes: particle size optimisation and compositional exploration.

Recently, layered rare-earth hydroxides (LRHs) have received growing attention in the field of theranostics. We have previously reported the hydrothermal synthesis of layered terbium hydroxide (LTbH), which exhibited high biocompatibility, reversible uptake of a range of model drugs, and release-sensitive phosphorescence. Despite these favourable properties, LTbH particles produced by the reported method suffered from poor size-uniformity (670 ± 564 nm), and are thus not suitable for therapeutic applications. To ameliorate this issue, we first derive an optimised hydrothermal synthesis method to generate LTbH particles with a high degree of homogeneity and reproducibility, within a size range appropriate for in vivo applications (152 ± 59 nm, n = 6). Subsequently, we apply this optimised method to synthesise a selected range of LRH materials (R = Pr, Nd, Gd, Dy, Er, Yb), four of which produced particles with an average size under 200 nm (Pr, Nd, Gd, and Dy) without the need for further optimisation. Finally, we incorporate Gd and Tb into LRHs in varying molar ratios (1 : 3, 1 : 1, and 3 : 1) and assess the combined magnetic relaxivity and phosphorescence properties of the resultant LRH materials. The lead formulation, LGd1.41Tb0.59H, was demonstrated to significantly shorten the T2 relaxation time of water (r2 = 52.06 mM-1 s-1), in addition to exhibiting a strong phosphorescence signal (over twice that of the other LRH formulations, including previously reported LTbH), therefore holding great promise as a potential multi-modal medical imaging probe.

X-Ray Visible Protein Scaffolds by Bulk Iodination.

Protein-based biomaterial use is expanding within medicine, together with the demand to visualize their placement and behavior in vivo. However, current medical imaging techniques struggle to differentiate between protein-based implants and surrounding tissue. Here a fast, simple, and translational solution for tracking transplanted protein-based scaffolds is presented using X-ray CT-facilitating long-term, non-invasive, and high-resolution imaging. X-ray visible scaffolds are engineered by selectively iodinating tyrosine residues under mild conditions using readily available reagents. To illustrate translatability, a clinically approved hernia repair mesh (based on decellularized porcine dermis) is labeled, preserving morphological and mechanical properties. In a mouse model of mesh implantation, implants retain marked X-ray contrast up to 3 months, together with an unchanged degradation rate and inflammatory response. The technique's compatibility is demonstrated with a range of therapeutically relevant protein formats including bovine, porcine, and jellyfish collagen, as well as silk sutures, enabling a wide range of surgical and regenerative medicine uses. This solution tackles the challenge of visualizing implanted protein-based biomaterials, which conventional imaging methods fail to differentiate from endogenous tissue. This will address previously unanswered questions regarding the accuracy of implantation, degradation rate, migration, and structural integrity, thereby accelerating optimization and safe translation of therapeutic biomaterials.

High levels of circulating epinephrine trigger apical cardiodepression in a ß2-adrenergic receptor/Gi-dependent manner: a new model of Takotsubo cardiomyopathy.

BACKGROUND: Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic ß(2)-adrenergic receptor (ß(2)AR) from canonical stimulatory G-protein-activated cardiostimulant to inhibitory G-protein-activated cardiodepressant pathways. METHODS AND RESULTS: We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via G(i) inactivation by pertussis toxin pretreatment. ß(2)AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a ß(2)AR-Gi-dependent manner. Preventing epinephrine-G(i) effects increased mortality in the Takotsubo model, whereas ß-blockers that activate ß(2)AR-G(i) exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality. CONCLUSIONS: We suggest that biased agonism of epinephrine for ß(2)AR-G(s) at low concentrations and for G(i) at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in ß(2)ARs explaining the differential regional responses. We suggest this epinephrine-specific ß(2)AR-G(i) signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.

Diaphragm rescue alone prevents heart dysfunction in dystrophic mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused, in most cases, by the complete absence of the 427 kDa cytoskeletal protein, dystrophin. There is no effective treatment, and affected individuals die from respiratory failure and cardiomyopathy by age 30. Here, we investigated whether cardiomyopathy could be prevented in animal models of DMD by increasing diaphragm utrophin or dystrophin expression and thereby restoring diaphragm function. In a transgenic mdx mouse, where utrophin was over expressed in the skeletal muscle and the diaphragm, but not in the heart, we found cardiac function, specifically right and left ventricular ejection fraction as measured using in vivo magnetic resonance imaging, was restored to wild-type levels. In mdx mice treated with a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that resulted in high levels of dystrophin restoration in the skeletal muscle and the diaphragm only, cardiac function was also restored to wild-type levels. In dystrophin/utrophin-deficient double-knockout (dKO) mice, a more severely affected animal model of DMD, treatment with a PPMO again produced high levels of dystrophin only in the skeletal muscle and the diaphragm, and once more restored cardiac function to wild-type levels. In the dKO mouse, there was no difference in heart function between treatment of the diaphragm plus the heart and treatment of the diaphragm alone. Restoration of diaphragm and other respiratory muscle function, irrespective of the method used, was sufficient to prevent cardiomyopathy in dystrophic mice. This novel mechanism of treating respiratory muscles to prevent cardiomyopathy in dystrophic mice warrants further investigation for its implications on the need to directly treat the heart in DMD.

Metabolic imaging of acute and chronic infarction in the perfused rat heart using hyperpolarised [1-13C]pyruvate.

Hyperpolarised (13)C MRI can be used to generate metabolic images of the heart in vivo. However, there have been no similar studies performed in the isolated perfused heart. Therefore, the aim of this study was to develop a method for the creation of (13)C metabolite maps of the perfused rat heart and to demonstrate the technique in a study of acute and chronic myocardial infarction. Male Wistar rat hearts were isolated, perfused and imaged before and after occlusion of the left anterior descending (LAD) coronary artery, creating an acute infarct group. In addition, a chronic infarct group was generated from hearts which had their LAD coronary artery occluded in vivo. Four weeks later, hearts were excised, perfused and imaged to generate metabolic maps of infused pyruvate and its metabolites lactate and bicarbonate. Myocardial perfusion and energetics were assessed by first-pass perfusion imaging and (31)P MRS, respectively. In both acute and chronically infarcted hearts, perfusion was reduced to the infarct region, as revealed by reduced gadolinium influx and lower signal intensity in the hyperpolarised pyruvate images. In the acute infarct region, there were significant alterations in the lactate (increased) and bicarbonate (decreased) signal ratios. In the chronically infarcted region, there was a significant reduction in both bicarbonate and lactate signals. (31)P-derived energetics revealed a significant decrease between control and chronic infarcted hearts. Significant decreases in contractile function between control and both acute and chronic infracted hearts were also seen. In conclusion, we have demonstrated that hyperpolarised pyruvate can detect reduced perfusion in the rat heart following both acute and chronic infarction. Changes in lactate and bicarbonate ratios indicate increased anaerobic metabolism in the acute infarct, which is not observed in the chronic infarct. Thus, this study has successfully demonstrated a novel imaging approach to assess altered metabolism in the isolated perfused rat heart.

Cellular coupling in the heart.

Fibroblasts in scar tissue elicit myocyte excitation and promote arrhythmia in mouse hearts.

Hybrid Polypyrrole and Polydopamine Nanosheets for Precise Raman/Photoacoustic Imaging and Photothermal Therapy.

The development of near-infrared light responsive conductive polymers provides a useful theranostic platform for malignant tumors by maximizing spatial resolution with deep tissue penetration for diagnosis and photothermal therapy. Herein, the self-assembly of ultrathin 2D polypyrrole nanosheets utilizing dopamine as a capping agent and a monolayer of octadecylamine as a template is demonstrated. The 2D polypyrrole-polydopamine nanostructure has tunable size distribution which shows strong absorption in the first and second near-infrared windows, enabling photoacoustic imaging and photothermal therapy. The hybrid double-layer is demonstrated to increase Raman intensity for 3D Raman imaging (up to two orders of magnitude enhancement and spatial resolution up to 1 µm). The acidic environment drives reversible doping of polypyrrole, which can be detected by Raman spectroscopy. The combined properties of the nanosheets can substantially enhance performance in dual-mode Raman and photoacoustic guided photothermal therapy, as shown by the 69% light to heat conversion efficiency and higher cytotoxicity against cancer spheroids. These pH-responsive features highlight the potential of 2D conductive polymers for applications in accurate, highly efficient theranostics.