The role of industry in advancing xenotransplantation.

PURPOSE OF REVIEW: Xenotransplantation offers the opportunity to alleviate the imbalance between the demand of patients with end stage organ failure and the supply of organs available for transplantation but remains aspirational. This review highlights how collaboration between academia and industry are essential for success. RECENT FINDINGS: The science of xenotransplantation has accelerated in recent years with key discoveries in genetic engineering, enabling disruption of genes facilitating rejection, and transgenic expression of desired human genes. Combined with similar progress directed toward induction of transplant tolerance, the stage has been set for meaningful progress. These advances are reviewed in detail elsewhere in this volume and argue that the breakthroughs needed to deliver substantial cross-species organ survival have largely been achieved, heralding a liminal stage of human xenotransplantation. However, xenotransplantation as a meaningful therapy for medically refractory end organ failure will not be realized through scientific innovation alone. The advent of broadly available, therapeutic xenogeneic tissues requires extensive development and regulatory expertise; the biotechnology/pharmaceutical industry can provide extensive resources and expertise in those essential areas. SUMMARY: Successful delivery of xenotransplantation as an available therapy for curing end stage organ failure is best accomplished through partnership and collaboration between academia and industry.

Differential activation of natriuretic peptide receptors modulates cardiomyocyte proliferation during development.

Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways.

Mutation in the transcriptional coactivator EYA4 causes dilated cardiomyopathy and sensorineural hearing loss.

We identified a human mutation that causes dilated cardiomyopathy and heart failure preceded by sensorineural hearing loss (SNHL). Unlike previously described mutations causing dilated cardiomyopathy that affect structural proteins, this mutation deletes 4,846 bp of the human transcriptional coactivator gene EYA4. To elucidate the roles of eya4 in heart function, we studied zebrafish embryos injected with antisense morpholino oligonucleotides. Attenuated eya4 transcript levels produced morphologic and hemodynamic features of heart failure. To determine why previously described mutated EYA4 alleles cause SNHL without heart disease, we examined biochemical interactions of mutant Eya4 peptides. Eya4 peptides associated with SNHL, but not the shortened 193-amino acid peptide associated with dilated cardiomyopathy and SNHL, bound wild-type Eya4 and associated with Six proteins. These data define unrecognized and crucial roles for Eya4-Six-mediated transcriptional regulation in normal heart function.

Hypertensive Emergency Team-Based Learning.

Audience: The target audiences for this team-based learning (TBL) activity are resident physicians and medical students. Introduction: According to the Centers for Disease Control and Prevention (CDC), nearly half of the adults in the United States have hypertension,1 which is a leading cause of cardiovascular disease and premature death.2 In extreme cases, patients may present in hypertensive emergencies, defined as an acute, marked elevation of systolic blood pressure >180mmHg or diastolic blood pressure >120mmHg with evidence of organ dysfunction.3,4 Patients presenting to the emergency department (ED) with symptoms of hypertensive emergencies must be promptly diagnosed and treated to prevent further morbidity and mortality. This TBL utilizes four clinical cases to educate resident physicians and medical students not only on the recognition of hypertensive emergencies, but also on the workup, management, and disposition of patients who present to the ED with hypertension. Educational Objectives: By the end of this TBL session, learners should be able to: 1) define features of asymptomatic hypertension versus hypertensive emergency, 2) discuss which patients with elevated blood pressure may require further diagnostic workup and intervention, 3) identify a differential diagnosis for patients presenting with elevated blood pressures, 4) recognize the features of different types of end-organ damage, 5) review an algorithm for the pharmacologic management of hypertensive emergencies, 6) indicate dosing and routes of various anti-hypertensive medications, 7) choose the appropriate treatment for a patient who is hypertensive and presenting with flash pulmonary edema, 8) identify an aortic dissection on computed tomography (CT), 9) choose the appropriate treatment for a patient who is hypertensive and presenting with an aortic dissection, 10) identify intracranial hemorrhage on CT, 11) choose the appropriate treatment for a patient who is hypertensive and presenting with an intracranial hemorrhage, and 12) describe the intervention for warfarin reversal. Educational Methods: This is a classic TBL that includes an individual readiness assessment test (iRAT), a multiple-choice group readiness assessment test (gRAT), and a group application exercise (GAE). Research Methods: Learners and instructors were given the opportunity to provide verbal feedback after completion of the TBL. Learners included senior medical students and first-, second-, and third-year emergency-medicine residents. Learners were specifically asked if they felt the cases were educational, relevant, and useful to their training. Results: Six resident physicians and three medical students volunteered their verbal feedback, and agreed when they were specifically asked if the cases were educational, relevant, and useful to their training. The same learners also agreed when asked if they felt the TBL was a more enjoyable activity than a direct lecture to refresh their knowledge and skills. One instructor observed that interns and medical students were generally able to reach a correct diagnosis; however, they seemed to struggle more with describing appropriate pharmacologic interventions when compared to more senior learners. Discussion: Hypertension is a common complaint and incidental finding in patients presenting to the ED. Given its non-specific value, it can be a difficult topic for the novice healthcare provider to master. The differential diagnosis for a patient presenting with hypertension is vast, ranging from benign to emergent, and can sometimes necessitate minimal to substantial workups. Thus, this TBL is a useful, relevant, and effective exercise for residents-in-training to review and understand the management of hypertension. Topics: Hypertension, hypertensive emergency, asymptomatic hypertension, flash pulmonary edema, aortic dissection, intracranial hemorrhage, warfarin reversal, team-based learning.

Klf2 is an essential regulator of vascular hemodynamic forces in vivo.

Hemodynamic responses that control blood pressure and the distribution of blood flow to different organs are essential for survival. Shear forces generated by blood flow regulate hemodynamic responses, but the molecular and genetic basis for such regulation is not known. The transcription factor KLF2 is activated by fluid shear stress in cultured endothelial cells, where it regulates a large number of vasoactive endothelial genes. Here, we show that Klf2 expression during development mirrors the rise of fluid shear forces, and that endothelial loss of Klf2 results in lethal embryonic heart failure due to a high-cardiac-output state. Klf2 deficiency does not result in anemia or structural vascular defects, and it can be rescued by administration of phenylephrine, a catecholamine that raises vessel tone. These findings identify Klf2 as an essential hemodynamic regulator in vivo and suggest that hemodynamic regulation in response to fluid shear stress is required for cardiovascular development and function.

High-resolution cardiovascular function confirms functional orthology of myocardial contractility pathways in zebrafish.

Phenotype-driven screens in larval zebrafish have transformed our understanding of the molecular basis of cardiovascular development. Screens to define the genetic determinants of physiological phenotypes have been slow to materialize as a result of the limited number of validated in vivo assays with relevant dynamic range. To enable rigorous assessment of cardiovascular physiology in living zebrafish embryos, we developed a suite of software tools for the analysis of high-speed video microscopic images and validated these, using established cardiomyopathy models in zebrafish as well as modulation of the nitric oxide (NO) pathway. Quantitative analysis in wild-type fish exposed to NO or in a zebrafish model of dilated cardiomyopathy demonstrated that these tools detect significant differences in ventricular chamber size, ventricular performance, and aortic flow velocity in zebrafish embryos across a large dynamic range. These methods also were able to establish the effects of the classic pharmacological agents isoproterenol, ouabain, and verapamil on cardiovascular physiology in zebrafish embryos. Sequence conservation between zebrafish and mammals of key amino acids in the pharmacological targets of these agents correlated with the functional orthology of the physiological response. These data provide evidence that the quantitative evaluation of subtle physiological differences in zebrafish can be accomplished at a resolution and with a dynamic range comparable to those achieved in mammals and provides a mechanism for genetic and small-molecule dissection of functional pathways in this model organism.

Heart failure and pulmonary hypertension.

When pulmonary hypertension (PH) and right ventricular dysfunction accompany heart failure, the impact on functional capacity and prognosis are ominous. Newer clinical strategies to preferentially lower pulmonary pressures and pulmonary vascular tone improve functional performance and symptoms of heart failure by targeting the nitric oxide signal transduction pathways, as with PDE5 inhibition. Additional studies are needed to determine if these therapies will impact long-term patient outcomes and elucidate the specific mechanisms whereby these treatments are effective. Furthermore, the recent finding that mutations in BMPR2 cause familial forms of pulmonary arterial hypertension and that BMPR2 expression is decreased in secondary forms of PH strongly implicate BMP signaling in the underlying pathophysiology of PH. Translation of emerging basic science insights in the vascular biology of PH and BMP signaling will provide novel therapeutic strategies for the spectrum of pulmonary hypertensive diseases.

Sildenafil improves exercise hemodynamics and oxygen uptake in patients with systolic heart failure.

BACKGROUND: Heart failure (HF) is frequently associated with dysregulation of nitric oxide-mediated pulmonary vascular tone. Sildenafil, a type 5 phosphodiesterase inhibitor, lowers pulmonary vascular resistance in pulmonary hypertension by augmenting intracellular levels of the nitric oxide second messenger, cyclic GMP. We tested the hypothesis that a single oral dose of sildenafil (50 mg) would improve exercise capacity and exercise hemodynamics in patients with chronic systolic HF through pulmonary vasodilation. METHODS AND RESULTS: Thirteen patients with New York Heart Association class III HF underwent assessment of right heart hemodynamics, gas exchange, and first-pass radionuclide ventriculography at rest and with cycle ergometry before and 60 minutes after administration of 50 mg of oral sildenafil. Sildenafil reduced resting pulmonary arterial pressure, systemic vascular resistance, and pulmonary vascular resistance, and increased resting and exercise cardiac index (P<0.05 for all) without altering mean arterial pressure, heart rate, or pulmonary capillary wedge pressure. Sildenafil reduced exercise pulmonary arterial pressure, pulmonary vascular resistance, and pulmonary vascular resistance/systemic vascular resistance ratio, which indicates a selective pulmonary vasodilator effect with exercise. Peak VO2 increased (15+/-9%) and ventilatory response to CO2 output (VE/VCO2 slope) decreased (16+/-5%) after sildenafil treatment. Improvements in right heart hemodynamics and exercise capacity were confined to patients with secondary pulmonary hypertension (rest pulmonary arterial pressure >25 mm Hg). CONCLUSIONS: The present study shows that in patients with systolic HF, type 5 phosphodiesterase inhibition with sildenafil improves peak VO2, reduces VE/VCO2 slope, and acts as a selective pulmonary vasodilator during rest and exercise in patients with HF and pulmonary hypertension.

Human-zebrafish non-coding conserved elements act in vivo to regulate transcription.

Whole genome comparisons of distantly related species effectively predict biologically important sequences--core genes and cis-acting regulatory elements (REs)--but require experimentation to verify biological activity. To examine the efficacy of comparative genomics in identification of active REs from anonymous, non-coding (NC) sequences, we generated a novel alignment of the human and draft zebrafish genomes, and contrasted this set to existing human and fugu datasets. We tested the transcriptional regulatory potential of candidate sequences using two in vivo assays. Strict selection of non-genic elements which are deeply conserved in vertebrate evolution identifies 1744 core vertebrate REs in human and two fish genomes. We tested 16 elements in vivo for cis-acting gene regulatory properties using zebrafish transient transgenesis and found that 10 (63%) strongly modulate tissue-specific expression of a green fluorescent protein reporter vector. We also report a novel quantitative enhancer assay with potential for increased throughput based on normalized luciferase activity in vivo. This complementary system identified 11 (69%; including 9 of 10 GFP-confirmed elements) with cis-acting function. Together, these data support the utility of comparative genomics of distantly related vertebrate species to identify REs and provide a scaleable, in vivo quantitative assay to define functional activity of candidate REs.

Locus for atrial fibrillation maps to chromosome 6q14-16.

BACKGROUND: Atrial fibrillation (AF), the most common clinical arrhythmia, is a major cause of morbidity and mortality. Although AF is often associated with other cardiovascular conditions, many patients present without an obvious etiology. Inherited forms of AF exist, but the causative gene has been defined only in a single family. We have identified a large family (family FAF-1) in which AF segregates as a Mendelian trait. METHODS AND RESULTS: Thirty-four family members were evaluated by 12-lead ECG, echocardiogram, 24-hour Holter monitoring, and laboratory studies. Individuals with electrocardiographically documented AF were defined as affected. Subjects were considered unaffected if they were >60 years of age, had no personal history of AF, and had no offspring with a history of AF. DNA was extracted and genotypic analyses were performed using polymorphic microsatellite markers. Evidence of linkage was obtained on chromosome 6, with a peak 2-point logarithm of the odds (LOD) score of 3.63 (theta=0) at the marker D6S1021. A maximal multipoint LOD score of 4.9 was obtained between D6S286 and D6S1021, indicating odds of approximately 100 000:1 in favor of this interval as the location of the gene defect responsible for AF in this family. The LOD scores were robust to changes in penetrance and allele frequency. Haplotype analyses further supported this minimal genetic interval. CONCLUSIONS: We have mapped a novel locus for AF to chromosome 6q14-16. The identification of the causative gene in this interval will be an important step in understanding the fundamental mechanisms of AF.

Hemodynamic effects of inhaled nitric oxide in right ventricular myocardial infarction and cardiogenic shock.

OBJECTIVES: We sought to determine whether or not inhaled nitric oxide (NO) could improve hemodynamic function in patients with right ventricular myocardial infarction (RVMI) and cardiogenic shock (CS). BACKGROUND: Inhaled NO is a selective pulmonary vasodilator that can decrease right ventricular afterload. METHODS: Thirteen patients (7 males and 6 females, age 65 +/- 3 years) presenting with electrocardiographic, echocardiographic, and hemodynamic evidence of acute inferior myocardial infarction associated with RVMI and CS were studied. After administration of supplemental oxygen (inspired oxygen fraction [F(i)O(2)] = 1.0), hemodynamic measurements were recorded before, during inhalation of NO (80 ppm at F(i)O(2) = 0.90) for 10 min, and 10 min after NO inhalation was discontinued (F(i)O(2) = 1.0). RESULTS: Breathing NO decreased the mean right atrial pressure by 12 +/- 3%, mean pulmonary arterial pressure by 13 +/- 2%, and pulmonary vascular resistance by 36 +/- 8% (all p < 0.05). Nitric oxide inhalation increased the cardiac index by 24 +/- 11% and the stroke volume index by 23 +/- 12% (p < 0.05). The NO administration did not change systemic arterial or pulmonary capillary wedge pressures. Contrast echocardiography identified three patients with a patent foramen ovale and right-to-left shunt flow while breathing at F(i)O(2) = 1.0. Breathing NO decreased shunt flow by 56 +/- 5% (p < 0.05) and was associated with markedly improved systemic oxygen saturation. CONCLUSIONS: Nitric oxide inhalation results in acute hemodynamic improvement when administered to patients with RVMI and CS.

Pharmacological HIF2α inhibition improves VHL disease-associated phenotypes in zebrafish model.

Patients with a germline mutation in von Hippel-Lindau (VHL) develop renal cell cancers and hypervascular tumors of the brain, adrenal glands, and pancreas as well as erythrocytosis. These phenotypes are driven by aberrant expression of HIF2α, which induces expression of genes involved in cell proliferation, angiogenesis, and red blood cell production. Currently, there are no effective treatments available for VHL disease. Here, using an animal model of VHL, we report a marked improvement of VHL-associated phenotypes following treatment with HIF2α inhibitors. Inactivation of vhl in zebrafish led to constitutive activation of HIF2α orthologs and modeled several aspects of the human disease, including erythrocytosis, pathologic angiogenesis in the brain and retina, and aberrant kidney and liver proliferation. Treatment of vhl(-/-) mutant embryos with HIF2α-specific inhibitors downregulated Hif target gene expression in a dose-dependent manner, improved abnormal hematopoiesis, and substantially suppressed erythrocytosis and angiogenic sprouting. Moreover, pharmacologic inhibition of HIF2α reversed the compromised cardiac contractility of vhl(-/-) embryos and partially rescued early lethality. This study demonstrates that small-molecule targeting of HIF2α improves VHL-related phenotypes in a vertebrate animal model and supports further exploration of this strategy for treating VHL disease.

Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase.

Doxorubicin is a highly effective anticancer chemotherapy agent, but its use is limited by its cardiotoxicity. To develop a drug that prevents this toxicity, we established a doxorubicin-induced cardiomyopathy model in zebrafish that recapitulates the cardiomyocyte apoptosis and contractility decline observed in patients. Using this model, we screened 3000 compounds and found that visnagin (VIS) and diphenylurea (DPU) rescue the cardiac performance and circulatory defects caused by doxorubicin in zebrafish. VIS and DPU reduced doxorubicin-induced apoptosis in cultured cardiomyocytes and in vivo in zebrafish and mouse hearts. VIS treatment improved cardiac contractility in doxorubicin-treated mice. Further, VIS and DPU did not reduce the chemotherapeutic efficacy of doxorubicin in several cultured tumor lines or in zebrafish and mouse xenograft models. Using affinity chromatography, we found that VIS binds to mitochondrial malate dehydrogenase (MDH2), a key enzyme in the tricarboxylic acid cycle. As with VIS, treatment with the MDH2 inhibitors mebendazole, thyroxine, and iodine prevented doxorubicin cardiotoxicity, as did treatment with malate itself, suggesting that modulation of MDH2 activity is responsible for VIS' cardioprotective effects. Thus, VIS and DPU are potent cardioprotective compounds, and MDH2 is a previously undescribed, druggable target for doxorubicin-induced cardiomyopathy.

Overexpression of human amyloidogenic light chains causes heart failure in embryonic zebrafish: a preliminary report.

AL cardiomyopathy leading to heart failure (HF) represents a significant cause of morbidity and mortality in systemic amyloidosis. However, the paucity of robust in vivo models of AL-induced cardiac dysfunction has limited our ability to probe the mechanisms of AL heart disease. To address this problem, we have developed a model of AL HF in zebrafish embryos by injection of in vitro transcribed mRNA encoding amyloidogenic light chain (aLC) into fertilized oocytes. We demonstrate that expression of aLC causes cardiomyopathy in developing zebrafish without significantly impairing extracardiac development. The cardiac ventricle of embryos expressing aLC exhibit impaired contractility, smaller size, and increased myocardial thickness which result in congestion and edema, features paralleling the clinical manifestations of amyloid cardiomyopathy. Phosphorylated p38, a marker of oxidative stress, was increased in response to aLC expression. No evidence of amyloid fibril deposition was identified. Thus, expression of aLC mRNA in zebrafish results in cardio toxic effects without fibril deposition. This is consistent with prior evidence indicating that aLC oligomers mediate cardiac dysfunction in vitro. This model will allow exploration of amyloid pathophysiology and testing of interventions to reduce and reverse the deleterious effects of amyloidosis on myocardial function.

STARS is essential to maintain cardiac development and function in vivo via a SRF pathway.

BACKGROUND: STARS (STriated muscle Activator of Rho Signaling) is a sarcomeric protein expressed early in cardiac development that acts as an acute stress sensor for pathological remodeling. However the role of STARS in cardiac development and function is incompletely understood. Here, we investigated the role of STARS in heart development and function in the zebrafish model and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: Expression of zebrafish STARS (zSTARS) first occurs in the somites by the 16 somite stage [17 hours post fertilization (hpf)]. zSTARS is expressed in both chambers of the heart by 48 hpf, and also in the developing brain, jaw structures and pectoral fins. Morpholino-induced knockdown of zSTARS alters atrial and ventricular dimensions and decreases ventricular fractional shortening (measured by high-speed video microscopy), with pericardial edema and decreased or absent circulation [abnormal cardiac phenotypes in 126/164 (77%) of morpholino-injected embryos vs. 0/152 (0%) of control morpholino embryos]. Co-injection of zsrf (serum response factor) mRNA rescues the cardiac phenotype of zSTARS knockdown, resulting in improved fractional shortening and ventricular end-diastolic dimensions. Ectopic over-expression of STARS in vitro activates the STARS proximal promoter, which contains a conserved SRF site. Chromatin immunoprecipitation demonstrates that SRF binds to this site in vivo and the SRF inhibitor CCG-1423 completely blocks STARS proximal reporter activity in H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study demonstrates for the first time that STARS deficiency severely disrupts cardiac development and function in vivo and revealed a novel STARS-SRF feed-forward autoregulatory loop that could play an essential role in STARS regulation and cardiac function.

Use of amino-terminal pro-B-type natriuretic peptide to guide outpatient therapy of patients with chronic left ventricular systolic dysfunction.

OBJECTIVES: The aim of this study was to evaluate whether chronic heart failure (HF) therapy guided by concentrations of amino-terminal pro-B-type natriuretic peptide (NT-proBNP) is superior to standard of care (SOC) management. BACKGROUND: It is unclear whether standard HF treatment plus a goal of reducing NT-proBNP concentrations improves outcomes compared with standard management alone. METHODS: In a prospective single-center trial, 151 subjects with HF due to left ventricular (LV) systolic dysfunction were randomized to receive either standard HF care plus a goal to reduce NT-proBNP concentrations ≤1,000 pg/ml or SOC management. The primary endpoint was total cardiovascular events between groups compared using generalized estimating equations. Secondary endpoints included effects of NT-proBNP-guided care on patient quality of life as well as cardiac structure and function, assessed with echocardiography. RESULTS: Through a mean follow-up period of 10 ± 3 months, a significant reduction in the primary endpoint of total cardiovascular events was seen in the NT-proBNP arm compared with SOC (58 events vs. 100 events, p = 0.009; logistic odds for events 0.44, p = 0.02); Kaplan-Meier curves demonstrated significant differences in time to first event, favoring NT-proBNP-guided care (p = 0.03). No age interaction was found, with elderly patients benefitting similarly from NT-proBNP-guided care as younger subjects. Compared with SOC, NT-proBNP-guided patients had greater improvements in quality of life, demonstrated greater relative improvements in LV ejection fraction, and had more significant improvements in both LV end-systolic and -diastolic volume indexes. CONCLUSIONS: In patients with HF due to LV systolic dysfunction, NT-proBNP-guided therapy was superior to SOC, with reduced event rates, improved quality of life, and favorable effects on cardiac remodeling. (Use of NT-proBNP Testing to Guide Heart Failure Therapy in the Outpatient Setting; NCT00351390).

Cardiac transplantation followed by dose-intensive melphalan and autologous stem-cell transplantation for light chain amyloidosis and heart failure.

BACKGROUND: Patients with light chain (AL) amyloidosis who present with severe heart failure due to cardiac involvement rarely survive more than 6 months. Survival after cardiac transplantation is markedly reduced due to the progression of amyloidosis. Autologous stem-cell transplantation (ASCT) has become a common therapy for AL amyloidosis, but there is an exceedingly high treatment-related mortality in patients with heart failure. METHODS: We developed a treatment strategy of cardiac transplant followed by ASCT. Twenty-six patients were evaluated, and of 18 eligible patients, nine patients underwent cardiac transplantation. Eight of these patients subsequently received an ASCT. RESULTS: Six of seven evaluable patients achieved a complete hematologic remission, and one achieved a partial remission. At a median follow-up of 56 months from cardiac transplant, five of seven patients are alive without recurrent amyloidosis. Their survival is comparable with 17,389 patients who received heart transplants for nonamyloid heart disease: 64% in nonamyloid vs. 60% in amyloid patients at 7 years (P=0.83). Seven of eight transplanted patients have had no evidence of amyloid in their cardiac allograft. CONCLUSIONS: This demonstrates that cardiac transplantation followed by ASCT is feasible in selected patients with AL amyloidosis and heart failure, and that such a strategy may lead to improved overall survival.

Eya4 regulation of Na+/K+-ATPase is required for sensory system development in zebrafish.

To investigate the mechanisms by which mutations in the human transcriptional co-activator EYA4 gene cause sensorineural hearing loss that can occur in association with dilated cardiomyopathy, we studied eya4 expression during zebrafish development and characterized eya4 deficiency. eya4 morphant fish embryos had reduced numbers of hair cells in the otic vesicle and lateral line neuromasts with impaired sensory responses. Analyses of candidate genes that are known to be expressed in a temporal and spatial pattern comparable to eya4 focused our analyses on atp1b2b, which encodes the beta2b subunit of the zebrafish Na+/K+-ATPase. We demonstrate atp1b2b levels are reduced in eya4 morphant fish and that morpholino oligonucleotides targeting the atp1b2b gene recapitulated the eya4 deficiency phenotypes, including heart failure, decreased sensory hair cell numbers in the otic vesicle and neuromasts, and abnormal sensory responses. Furthermore, atp1b2b overexpression rescued these phenotypes in eya4 morphant fish. We conclude that eya4 regulation of Na+/K+-ATPase is crucial for the development of mechanosensory cells and the maintenance of cardiac function in zebrafish.

Mutant desmocollin-2 causes arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically heterogeneous heart-muscle disorder characterized by progressive fibrofatty replacement of right ventricular myocardium and an increased risk of sudden cardiac death. Mutations in desmosomal proteins that cause ARVC have been previously described; therefore, we investigated 88 unrelated patients with the disorder for mutations in human desmosomal cadherin desmocollin-2 (DSC2). We identified a heterozygous splice-acceptor-site mutation in intron 5 (c.631-2A-->G) of the DSC2 gene, which led to the use of a cryptic splice-acceptor site and the creation of a downstream premature termination codon. Quantitative analysis of cardiac DSC2 expression in patient specimens revealed a marked reduction in the abundance of the mutant transcript. Morpholino knockdown in zebrafish embryos revealed a requirement for dsc2 in the establishment of the normal myocardial structure and function, with reduced desmosomal plaque area, loss of the desmosome extracellular electron-dense midlines, and associated myocardial contractility defects. These data identify DSC2 mutations as a cause of ARVC in humans and demonstrate that physiologic levels of DSC2 are crucial for normal cardiac desmosome formation, early cardiac morphogenesis, and cardiac function.

A novel locus for dilated cardiomyopathy, diffuse myocardial fibrosis, and sudden death on chromosome 10q25-26.

OBJECTIVES: We sought to identify the genetic locus for an inherited form of dilated cardiomyopathy (DCM) that is characterized by diffuse myocardial fibrosis and sudden death. BACKGROUND: Genetic studies have mapped multiple loci for DCM, which is a major cause of nonischemic heart failure; however, the genes responsible for the majority of cases have yet to be identified. METHODS: Sixty-six family members were evaluated by 12-lead electrocardiogram (ECG), echocardiogram, and laboratory studies. Individuals with echocardiographically documented DCM were defined as affected. Subjects were considered unaffected if they were older than 20 years of age, had a normal ECG and echocardiogram, no personal history of heart failure, and had no affected offspring. Genotyping was performed using polymorphic markers. RESULTS: Genome-wide linkage analysis identified a novel locus for this inherited phenotype on chromosome 10q25.3-q26.13. Peak two-point logarithm of the odds scores >3.0 were obtained independently with each family using the markers D10S1773 and D10S1483, respectively. Haplotype analyses defined a critical interval of 14.0 centiMorgans between D10S1237 and D10S1723, corresponding to a physical distance of 9.5 megabases. Multipoint linkage analyses confirmed this interval and generated a peak logarithm of the odds score of 8.2 indicating odds of >100,000,000:1 in favor of this interval as the location of the gene defect responsible for DCM in these families. CONCLUSIONS: We have mapped a novel locus for cardiomyopathy, diffuse myocardial fibrosis, and sudden death to chromosome 10q25-q26. The identification of the causative gene in this interval will be an important step in understanding the fundamental mechanisms of heart failure and sudden death.