Superselective endovascular tissue access using trans-vessel wall technique: feasibility study for treatment applications in heart, pancreas and kidney in swine.

OBJECTIVES: With the emergence of targeted cell transplantation and gene therapy, there is a need for minimally invasive tissue access to facilitate delivery of therapeutic substrate. The objective of this study was to demonstrate the suitability of an endovascular device which is able to directly access tissue and deliver therapeutic agent to the heart, kidney and pancreas without need to seal the penetration site. METHODS: In vivo experiments were performed in 30 swine, including subgroups with follow-up to evaluate complications. The previously described trans-vessel wall (VW) device was modified to be sharper and not require tip detachment to seal the VW. Injections into targets in the heart (n = 13, 24-h follow-up n = 5, 72-h follow-up n = 3), kidney (n = 8, 14-day follow-up n = 3) and pancreas (n = 5) were performed. Some animals were used for multiple organ injections. Follow-up consisted of clinical monitoring, angiography and necropsy. Transvenous (in heart) and transarterial approaches (in heart, kidney and pancreas) were used. Injections were targeted towards the subepicardium, endomyocardium, pancreas head and tail, and kidney subcapsular space and cortex. RESULTS: Injections were successful in target organs, visualized by intraparenchymal contrast on fluoroscopy and by necropsy. No serious complications (defined as heart failure or persistent arrhythmia, haemorrhage requiring treatment or acute kidney injury) were encountered over a total of 157 injections. CONCLUSIONS: The trans-VW device can achieve superselective injections to the heart, pancreas and kidney for delivery of therapeutic substances without tip detachment. All parts of these organs including the subepicardium, pancreas tail and renal subcapsular space can be efficiently reached.

Multipotent islet-1 cardiovascular progenitors in development and disease.

During the past several years, advances at the intersection of cardiovascular development and heart stem cell biology have begun to reshape our view of the fundamental logic that drives the formation of discrete tissue components in the mammalian heart. Although many of the critical genes that control cardiac myogenesis have been identified, our understanding of how a highly diverse and specialized subset of heart cell lineages arises from mesodermal precursors and is subsequently assembled into distinct muscle chambers, coronary arterial tree and large vessels, valvular tissue, and conduction system/pacemaker cells remains at a relatively primitive stage. Recent studies have uncovered a diverse group of closely related heart progenitors that are central in controlling and coordinating these complex steps of cardiogenesis. Understanding the pathways that control their formation, renewal, and subsequent conversion to specific differentiated progeny forms the underpinning for unraveling the pathways for congenital heart disease and has direct relevance to cardiovascular regenerative medicine. This current brief review highlights the discovery and delineation of the role of Islet-1 cardiovascular progenitors in the generation of diverse heart cell lineages and how the implications of these findings are revising our classification and thinking about congenital heart disease in general.

Reduced expression of the Sp4 gene in mice causes deficits in sensorimotor gating and memory associated with hippocampal vacuolization.

HF-1B/SP4:, a member of the Sp1 family of transcription factors, is expressed restrictively in the developing nervous system and most abundantly in adult hippocampus in mice. Here, we report the generation of hypomorphic Sp4 allele mice, in which the Sp4 deficiency can be rescued by the expression of Cre recombinase. Vacuolization was detected in the hippocampal gray matter of the mutant Sp4-deficient mice. Expression analysis of Sp4 mutant hippocampi revealed an age-dependent decrease in neurotrophin-3 expression in the dentate granule cells. Hypomorphic Sp4 mutant mice displayed robust deficits in both sensorimotor gating and contextual memory. The restoration of Sp4 expression, via a Cre-dependent rescue strategy, completely rescued all the observed molecular, histological and behavioral abnormalities. Our studies thus reveal a novel Sp4 pathway that is essential for hippocampal integrity and modulates behavioral processes relevant to psychiatric disorders.

In vivo high-efficiency transcoronary gene delivery and Cre-LoxP gene switching in the adult mouse heart.

High-efficiency somatic gene transfer in adult mouse heart has not yet been achieved in vivo. Here, we demonstrate high-efficiency in vivo transcoronary gene delivery to the adult murine myocardium using a catheter-based technique with recombinant adenovirus (AdV) and adeno-associated virus (AAV) vectors in normal and genetically engineered mice. The method involves immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic segmental injection of cardioplegic solution containing substance P and viral vectors. Gene expression measured using a LacZ marker gene was observed throughout both ventricles. The expression efficiency of a cytoplasmic LacZ marker gene in the left ventricular myocardium was 56.4+/-14.5% (mean+/-s.d.) at 4 days with an AdV vector, and with an AAV vector it was 81.0+/-5.9% at 4 weeks. Following AAV gene transfer, no gene expression was found in kidney, brain, lung, and spleen, but there was slight expression in liver. In addition, we demonstrate temporally controlled genetic manipulation in the heart with an efficiency of 54.6+/-5.2%, by transferring an AdV vector carrying Cre recombinase in ROSA26 flox-LacZ reporter mice. Procedure-related mortality was 16% for AdV and zero for AAV transfer. Thus, this method provides efficient, relatively homogeneous gene expression in both ventricles of the adult mouse heart, and offers a novel approach for conditional gene rescue or ablation in genetically engineered mouse models.

A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I(to) and confers susceptibility to ventricular tachycardia.

KChIP2, a gene encoding three auxiliary subunits of Kv4.2 and Kv4.3, is preferentially expressed in the adult heart, and its expression is downregulated in cardiac hypertrophy. Mice deficient for KChIP2 exhibit normal cardiac structure and function but display a prolonged elevation in the ST segment on the electrocardiogram. The KChIP2(-/-) mice are highly susceptible to the induction of cardiac arrhythmias. Single-cell analysis revealed a substrate for arrhythmogenesis, including a complete absence of transient outward potassium current, I(to), and a marked increase in action potential duration. These studies demonstrate that a defect in KChIP2 is sufficient to confer a marked genetic susceptibility to arrhythmias, establishing a novel genetic pathway for ventricular tachycardia via a loss of the transmural gradient of I(to).

Suppressor of cytokine signaling-3 is a biomechanical stress-inducible gene that suppresses gp130-mediated cardiac myocyte hypertrophy and survival pathways.

The gp130 cytokine receptor activates a cardiomyocyte survival pathway during the transition to heart failure following the biomechanical stress of pressure overload. Although gp130 activation is observed transiently during transverse aortic constriction (TAC), its mechanism of inactivation is largely unknown in cardiomyocytes. We show here that suppressor of cytokine signaling 3 (SOCS3), an intrinsic inhibitor of JAK, shows biphasic induction in response to TAC. The induction of SOCS3 was closely correlated with STAT3 phosphorylation, as well as the activation of an embryonic gene program, suggesting that cardiac gp130-JAK signaling is precisely controlled by this endogenous suppressor. In addition to its cytoprotective action, gp130-dependent signaling induces cardiomyocyte hypertrophy. Adenovirus-mediated gene transfer of SOCS3 to ventricular cardiomyocytes completely suppressed both hypertrophy and antiapoptotic phenotypes induced by leukemia inhibitory factor (LIF). To our knowledge, this is the first clear evidence that these two separate cardiomyocyte phenotypes induced by gp130 activation lie downstream of JAK. Three independent signaling pathways, STAT3, MEK1-ERK1/2, and AKT activation, that are coinduced by LIF stimulation were completely suppressed by SOCS3 overexpression. We conclude that SOCS3 is a mechanical stress-inducible gene in cardiac muscle cells and that it directly modulates stress-induced gp130 cytokine receptor signaling as the key molecular switch for a negative feedback circuit for both myocyte hypertrophy and survival.

Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Galphaq/Galpha11 in cardiomyocytes.

Myocardial hypertrophy is an adaptational response of the heart to increased work load, but it is also associated with a high risk of cardiac mortality due to its established role in the development of cardiac failure, one of the leading causes of death in developed countries. Multiple growth factors and various downstream signaling pathways involving, for example, ras, gp-130 (ref. 4), JNK/p38 (refs. 5,6) and calcineurin/NFAT/CaM-kinase have been implicated in the hypertrophic response. However, there is evidence that the initial phase in the development of myocardial hypertrophy involves the formation of cardiac para- and/or autocrine factors like endothelin-1, norepinephrine or angiotensin II (refs. 7,8), the receptors of which are coupled to G-proteins of the Gq/11-, G12/13- and Gi/o-families. Cardiomyocyte-specific transgenic overexpression of alpha1-adrenergic or angiotensin (AT1)-receptors as well as of the Gq alpha-subunit, Galphaq, results in myocardial hypertrophy. These data demonstrate that chronic activation of the Gq/G11-family is sufficient to induce myocardial hypertrophy. In order to test whether Gq/G11 mediate the physiological hypertrophy response to pressure overload, we generated a mouse line lacking both Galphaq and Galpha11 in cardiomyocytes. These mice showed no detectable ventricular hypertrophy in response to pressure-overload induced by aortic constriction. The complete lack of a hypertrophic response proves that the Gq/G11-mediated pathway is essential for cardiac hypertrophy induced by pressure overload and makes this signaling process an interesting target for interventions to prevent myocardial hypertrophy.

Dobutamine stress cine-MRI of cardiac function in the hearts of adult cardiomyocyte-specific VEGF knockout mice.

A mouse model of non-necrotic vascular deficiency in the adult heart was studied using cine-magnetic resonance imaging (MRI) and other techniques. The mice lacked cardiomyocyte-derived vascular endothelial growth factor (VEGF) following a targeted knockout in the ventricular cardiomyocytes. Quantitative endothelial labeling showed that the capillary density was significantly reduced in the hearts of knockout mice. Gene expression patterns suggested that they were hypoxic. Semiautomated MR image analysis was employed to obtain both global and regional measurements of left ventricular function at 10 or more time points through the cardiac cycle. MRI measurements showed a marked reduction in ejection fraction both at rest and under low- and high-dose dobutamine stress. Regional wall thickness, thickening, and displacement were all attenuated in the knockout mice. A prolonged high-dose dobutamine challenge was monitored by MRI. A maximal response was sustained for 90 minutes, suggesting that it did not depend on endogenous glycogen stores.

A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function.

The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to beta-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.

Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy.

Although cytoskeletal mutations are known causes of genetically based forms of dilated cardiomyopathy, the pathways that link these defects with cardiomyopathy are unclear. Here we report that the alpha-actinin-associated LIM protein (ALP; Alp in mice) has an essential role in the embryonic development of the right ventricular (RV) chamber during its exposure to high biomechanical workloads in utero. Disruption of the gene encoding Alp (Alp) is associated with RV chamber dilation and dysfunction, directly implicating alpha-actinin-associated proteins in the onset of cardiomyopathy. In vitro assays showed that Alp directly enhances the capacity of alpha-actinin to cross-link actin filaments, indicating that the loss of Alp function contributes to destabilization of actin anchorage sites in cardiac muscle. Alp also colocalizes at the intercalated disc with alpha-actinin and gamma-catenin, the latter being a known disease gene for human RV dysplasia. Taken together, these studies point to a novel developmental pathway for RV dilated cardiomyopathy via instability of alpha-actinin complexes.

Effects of deletion of muscle LIM protein on myocyte function.

Muscle LIM protein (MLP) may serve as a scaffold protein on the actin-based cytoskeleton, and mice deficient in this protein (MLPKO) have been recently reported to develop dilated cardiomyopathy. To determine the causes of depressed contractility in this model, we measured intracellular Ca2+ concentration ([Ca2+]i) transients (fluo 3), cell shortening, L-type Ca2+ channel current (I(Ca,L)), Na/Ca exchanger current (I(Na/Ca)), and sarcoplasmic reticulum (SR) Ca content in left ventricular MLPKO myocytes. I(Ca,L)-voltage relationships, I(Na/Ca) density, and membrane capacitance did not differ between wild-type (WT) and MLPKO myocytes. The peak systolic [Ca2+]i was significantly increased in MLPKO myocytes (603 +/- 54 vs. 349 +/- 18 nM in WT myocytes). The decline of [Ca2+]i transients was accelerated in MLPKO myocytes, and SR Ca2+ content was increased by 21%, indicating that SR Ca2+-ATPase function is normal or enhanced in MLPKO myocytes. Confocal imaging of actin filaments stained with tetramethylrhodamine isothiocyanate-labeled phalloidin showed disorganization of myofibrils and abnormal alignment of Z bands, and fractional shortening was significantly diminished in MLPKO myocytes compared with that in WT myocytes at comparable peak [Ca2+]i. Thus a reduced [Ca2+]-induced shortening may be involved in the pathogenesis of myocardial dysfunction in this genetic model of heart failure.

Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43.

Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1+/-0.13 versus 1.66+/-0.06; P:<0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.

[Developmental genes and heart disease]. / Genes del desarrollo y enfermedad cardíaca.

The past three years can be considered in cardiology as critical for understanding the relevance of developmental genes in the adult cardiac physiology. Also, for the first time, endogenous control of programmed cell death has been demonstrated to mark the transition between normal adaptation and cardiac hypertrophy. Most of this work has been based on previous analysis using molecular markers of cardiac determination and differentiation, work that has served a double aim: First, the determination of the cellular process that contribute to the specification of the working heart and secondly, the characterization of key regulatory factors in cardiogenesis. These studies in conjunction with the recent availability of single gene mutation in transgenic mice have furnished a new perspective in the nature of cardiac defects either in shape or function. Here we review some of the key factors in cardiac morphogenesis from the perspective of the analysis of gene mutation.

Genomic circuits and the integrative biology of cardiac diseases.

Human cardiac disease is the result of complex interactions between genetic susceptibility and environmental stress. The challenge is to identify modifiers of disease, and to design new therapeutic strategies to interrupt the underlying disease pathways. The availability of genomic databases for many species is uncovering networks of conserved cardiac-specific genes within given physiological pathways. A new classification of human cardiac diseases can be envisaged based on the disruption of integrated genomic circuits that control heart morphogenesis, myocyte survival, biomechanical stress responses, cardiac contractility and electrical conduction.

A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

Hepatocyte-specific mutation establishes retinoid X receptor alpha as a heterodimeric integrator of multiple physiological processes in the liver.

A large number of physiological processes in the adult liver are regulated by nuclear receptors that require heterodimerization with retinoid X receptors (RXRs). In this study, we have used cre-mediated recombination to disrupt the mouse RXRalpha gene specifically in hepatocytes. Although such mice are viable, molecular and biochemical parameters indicate that every one of the examined metabolic pathways in the liver (mediated by RXR heterodimerization with PPARalpha, CARbeta, PXR, LXR, and FXR) is compromised in the absence of RXRalpha. These data demonstrate the presence of a complex circuitry in which RXRalpha is integrated into a number of diverse physiological pathways as a common regulatory component of cholesterol, fatty acid, bile acid, steroid, and xenobiotic metabolism and homeostasis.

Differentiation of cardiomyocytes in floating embryoid bodies is comparable to fetal cardiomyocytes.

Embryonic stem cells will cluster and differentiate into embryoid bodies, which can develop spontaneous rhythmic contractions. From these embryoid bodies, cardiomyocytes can be isolated based on density by a discontinuous Percoll gradient. These cardiomyocytes differentiate into ventricular myocytes, which is demonstrated by the expression of the ventricular specific isoform of the myosin light chain 2 gene. In this study the functional expression of ion channels was compared between fetal cardiomyocytes (in vivo) and stem cell derived cardiomyocytes (in vitro). Sodium and calcium currents together with transient potassium currents could be detected in early developmental stages (