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Cardiac rhythms arise from electrical activity generated by precisely timed opening and closing of ion channels in individual cardiac myocytes. These impulses spread throughout the cardiac muscle to manifest as electrical waves in the whole heart. Regularity of electrical waves is critically important since they signal the heart muscle to contract, driving the primary function of the heart to act as a pump and deliver blood to the brain and vital organs. When electrical activity goes awry during a cardiac arrhythmia, the pump does not function, the brain does not receive oxygenated blood, and death ensues. For more than 50 years, mathematically based models of cardiac electrical activity have been used to improve understanding of basic mechanisms of normal and abnormal cardiac electrical function. Computer-based modeling approaches to understand cardiac activity are uniquely helpful because they allow for distillation of complex emergent behaviors into the key contributing components underlying them. Here we review the latest advances and novel concepts in the field as they relate to understanding the complex interplay between electrical, mechanical, structural, and genetic mechanisms during arrhythmia development at the level of ion channels, cells, and tissues. We also discuss the latest computational approaches to guiding arrhythmia therapy.

The presence of the blood-brain barrier complicates drug delivery in the development of therapeutic agents for the treatment of glioblastoma multiforme (GBM). The use of local gene transfer in the brain has the potential to overcome this delivery barrier by allowing the expression of therapeutic agents directly at the tumor site. In this study, we describe the development of a recombinant adeno-associated (rAAV) serotype 8 vector that encodes an optimized soluble inhibitor, termed sVEGFR1/R2, of vascular endothelial growth factor (VEGF). VEGF is an angiogenic factor highly up-regulated in GBM tumor tissue and correlates with disease progression. In subcutaneous models of GBM, VEGF inhibition following rAAV-mediated gene transfer significantly reduces overall tumor volume and increases median survival time following a single administration of vector. Using orthotopic brain tumor models of GBM, we find that direct intracranial administration of the rAAV-sVEGFR1/R2 vector to the tumor site demonstrates anti-tumor efficacy at doses that are not efficacious following systemic delivery of the vector. We propose that rAAV-mediated gene transfer of a potent soluble VEGF inhibitor in the CNS represents an effective antiangiogenic treatment strategy for GBM.

Reperfusion arrhythmias result from pathologies of cardiac myocyte physiology that develop when previously ischemic myocardium experiences a restoration of normal perfusion. The mechanisms of reperfusion arrhythmogenesis, which involve many components of a highly coupled nonlinear system, have been under investigation for many years. Despite these efforts, an effective therapy for the prevention of reperfusion arrhythmias has yet to be translated into routine clinical practice. Because of the highly complex nature of the problem, we have developed a cardiac cellular mathematical model tailored to the study of reperfusion arrhythmogenesis. This model allows more realistic simulations of ischemia and reperfusion than have been conducted previously, because it includes coupled intra- and extracellular pH regulation systems, as well as modification of the activity of ionic channels and exchangers secondary to changes in pH and the concentrations of ATP, ADP and other associated metabolites. We show that the model more closely reproduces experimental ischemia data than other existing models. Because of this, the model has strong promise for elucidating mechanisms of reperfusion arrhythmogenesis.

Object Many factors relating to the safety and efficacy of convection-enhanced delivery (CED) into intracranial tumors are poorly understood. To investigate these factors further and establish a more clinically relevant large animal model, with the potential to investigate CED in large, spontaneous tumors, the authors developed a magnetic resonance (MR) imaging–compatible system for CED of liposomal nanoparticles into the canine brain, incorporating real-time MR imaging. Additionally any possible toxicity of liposomes containing Gd and the chemotherapeutic agent irinotecan (CPT-11) was assessed following direct intraparenchymal delivery. Methods Four healthy laboratory dogs were infused with liposomes containing Gd, rhodamine, or CPT-11. Convection-enhanced delivery was monitored in real time by sequential MR imaging, and the volumes of distribution were calculated from MR images and histological sections. Assessment of any toxicity was based on clinical and histopathological evaluation. Convection-enhanced delivery resulted in robust volumes of distribution in both gray and white matter, and real-time MR imaging allowed accurate calculation of volumes and pathways of distribution. Results Infusion variability was greatest in the gray matter, and was associated with leakage into ventricular or subarachnoid spaces. Complications were minimal and included mild transient proprioceptive deficits, focal hemorrhage in 1 dog, and focal, mild perivascular, nonsuppurative encephalitis in 1 dog. Conclusions Convection-enhanced delivery of liposomal Gd/CPT-11 is associated with minimal adverse effects in a large animal model, and further assessment for use in clinical patients is warranted. Future studies investigating real-time monitored CED in spontaneous gliomas in canines are feasible and will provide a unique, clinically relevant large animal translational model for testing this and other therapeutic strategies.

Vascular endothelial growth factor (VEGF) is an important regulator of tumor angiogenesis and vascular permeability, and has been implicated both in progression of central nervous system (CNS) tumors and development of vasogenic peritumoral edema. A retrospective study was done to characterize the levels of expression of the 3 major canine VEGF isoforms (VEGF120, VEGF164, VEGF188) in a variety of spontaneous canine CNS tumors using quantitative TaqMan reverse transcription real-time polymerase chain reaction. Presence and degree of peritumoral edema also were determined in sampled tumors using magnetic resonance imaging (MRI). Increased expression of VEGF relative to normal cerebral cortex tissue was seen predominantly in high grade astrocytic (grade IV) and oligodendroglial (grade III) tumors, with lower expression in low grade astrocytomas (grade II) and meningiomas (grade I). All 3 major VEGF isoforms were present; VEGF164 was the predominant isoform, particularly in the tumors with the highest VEGF expression. Peritumoral edema was present in all tumor types; however, a significant association between the extent of peritumoral edema and the level of VEGF expression was not apparent.

In this study, recombinant AAV vectors pseudotyped with viral capsids derived from AAV serotypes 7 and 8 were evaluated for gene transfer in the murine striatum relative to vectors pseudotyped with AAV serotypes 2, 5, and 6. In comparison with rAAV serotype 2, pseudotyped vectors derived from AAV-7 and AAV-8 have increased transduction efficiency in the murine CNS, with the rank order rAAV-7 > rAAV-8 > rAAV-5 > rAAV-2 = rAAV-6, with all vectors demonstrating a marked tropism for neuronal transduction. Pseudotyped rAAV vector gene transfer in the brain after preimplantation of a murine 4C8 glioblastoma tumor was also evaluated. Efficiency of gene transfer to the orthotopic tumor was increased when using AAV-6, -7, and -8 capsid proteins in comparison with serotype 2, with the order rAAV-8 = rAAV-7 > rAAV-6 > rAAV-2 > rAAV-5. The increased gene transfer efficiency of rAAV vectors pseudotyped with the rAAV-8 capsid also provided enhanced therapeutic efficacy in a mouse model of glioblastoma multiforme, using vectors encoding an inhibitor of the vascular endothelial growth factor pathway. These studies demonstrate that rAAV vectors pseudotyped with capsids derived from AAV serotypes 7 and 8 provide enhanced gene transfer in the murine CNS and may offer increased therapeutic efficacy in the treatment of neurological disease.

Glioblastoma Multiforme (GBM) is the most common primary brain tumor leading to more than 12,000 deaths per year in the US. Radiation and chemotherapy has achieved limited success in the treatment of GBM and gene therapy may be a promising new strategy. We have investigated the gene transfer efficiencies of AAV serotypes –2, -5 and –6 encoding GFP in the murine CNS. AAV vectors derived from serotype 6 using the strong constitutive CAG promoter provide the optimum transduction efficiency in both the absence (normal brain) and presence of tumor (murine 4C8 glioma and human U87 glioma) in the murine brain. Following this work, the therapeutic potential of recombinant AAV-6 gene delivery of soluble forms of the human vascular endothelial growth factor receptors (sVEGFR) for blocking glioma growth in vivo following local (intra-tumoral) and systemic administration was investigated. Initially, soluble forms of VEGFR1 (domains 1-7) and VEGFR2 (domains 1-7) were constructed by fusion to an Fc segment of human IgG1, however, both receptors showed minimal efficacy in inhibiting subcutaneous C6 (rat glioma) and U87 glioma tumors following expression in vivo. To address this lack of efficacy, a chimeric soluble VEGF receptor was constructed, comprising of domains from VEGFR1 and VEGFR2 linked to IgG1 Fc, termed sVEGFR1/2. Assays in vitro and in CAM demonstrated that the sVEGFR1/2 molecule has VEGF binding affinities comparable to that of VEGFR1 coupled with the favorable bioavailability of the sVEGFR2 receptor. Furthermore, the sVEGFR1/2 molecule completely prevented tumor progression >80 days post-tumor implantation in comparison to the control group in a murine sub-cutaneous U87 human glioma model and demonstrated similar efficacy in the C6 model. The potency of a recombinant AAV-6 vector encoding the sVEGFR1/2 transgene to treat orthotopic murine 4C8 glial tumors in BFD2F1 immuno-competent mice was then examined. Animals received AAV-VEGFR1/2 delivered intra-tumorally by stereotaxic injection into pre-established (7-day) 4C8 tumors. Thirty days post-tumor implantation, animals treated with AAV6-VEGFR1/2 vector showed > 80% decrease in tumor growth in comparison to control animals as assessed by MRI. Survival was also significantly extended by treatment with the soluble chimeric VEGF receptor expressed from the AAV-6 vector. In addition, the efficacy of systemic sVEGFR1/2 expression following AAV mediated gene transfer to the liver on orthotopic 4C8 tumor growth was compared to local delivery to determine the impact of the blood brain barrier of the efficacy of the transgene. This work suggests that local delivery of a potent anti-VEGF agent by AAV-6 mediated gene transfer may be a novel and effective strategy for blocking the growth of human glioma tumors in vivo and may present a niche in which local gene therapy has advantage over systemic protein therapies.

Myocardial ischemia-reperfusion (IR) injury represents a constellation of pathological processes that occur when ischemic myocardium experiences a restoration of perfusion. Reentrant arrhythmias, which represent a particularly lethal manifestation of IR injury, can result when ischemic tissue exhibits decreased excitability and/or changes of action potential duration (APD), conditions that precipitate unidirectional conduction block. Many of the cellular components that are involved with IR injury are modulated by pH and/or phosphometabolites such as ATP and phosphocreatine (PCr), all of which can be manipulated in vivo and potentially in the clinical setting. Using a mathematical model of the cardiomyocyte that we previously developed to study ischemia and reperfusion, we performed a series of simulations with the aim of determining whether pH- or phosphometabolite-related processes play a more significant role in generating changes in excitability and action potential morphology that are associated with the development of reentry. In our simulations, persistent shortening of APD, action potential amplitude (APA), and depolarization of the resting membrane potential were more severe when ATP and PCr availability were suppressed during reperfusion than when extracellular pH recovery was inhibited. Reduced phosphometabolite availability and pH recovery affected multiple ion channels and exchangers. Some of these effects were the result of direct modulation by phosphometabolites and/or acidosis, while others resulted from elevated sodium and calcium loads during reperfusion. In addition, increasing ATP and PCr availability during reperfusion was more beneficial in terms of increasing APD and APA than was increasing the amount of pH recovery. Together, these results suggest that therapies directed at increasing ATP and/or PCr availability during reperfusion may be more beneficial than perturbing pH recovery with regard to mitigating action potential changes that increase the likelihood of reentrant arrhythmias.

Reperfusion injury results from pathologies of cardiac myocyte physiology that develop when previously ischemic myocardium experiences a restoration of normal perfusion. Events in the development of reperfusion injury begin with the restoration of a proton gradient upon reperfusion, which then allows the sodium-proton exchanger (NHE) to increase flux, removing protons from the intracellular space while importing sodium. The resulting sodium overload drives increased reverse-mode sodium-calcium exchanger (NCX) activity, creating a secondary calcium overload that has pathologic consequences. One of the attempts to reduce reperfusion-related damage, NHE inhibition, has shown little clinical benefit, and only when NHE inhibitors are given prior to reperfusion. In an effort to further understand why NHE inhibitors have been largely unsuccessful, we employed a new mathematical cardiomyocyte model that we developed for the study of ischemia and reperfusion. Using this model, we simulated 20 minutes of ischemia and 10 minutes of reperfusion, while also simulating NHE inhibition by reducing NHE flux in our model by varying amounts and at different time points. In our simulations, when NHE inhibition is applied at the onset of reperfusion, increasing the degree of inhibition increases the peak sodium and calcium concentrations, as well as reducing intracellular pH recovery. When inhibition was instituted at earlier time points, some modest improvements were seen, largely due to reduced sodium concentrations prior to reperfusion. Analysis of all sodium flux pathways suggests that the sodium-potassium pump (NaK) plays the largest role in exacerbated sodium overload during reperfusion, and that reduced NaK flux is largely the result of impaired pH recovery. While NHE inhibition does indeed reduce sodium influx through that exchanger, the resulting prolongation of intracellular acidosis paradoxically increases sodium overload, largely mediated by impaired NaK function., Author Summary Myocardial ischemia, commonly observed when arteries supplying the heart become occluded, results when cardiac tissue receives inadequate blood perfusion. In order to minimize the amount of cardiac damage, ischemic tissue must be reperfused. However, reperfusion can result in deleterious effects that leave the heart muscle sicker than if the ischemia had been allowed to continue. Examples of these reperfusion injuries include lethal arrhythmias and an increased region of cell death. Some of the early events that result in reperfusion injury include changes in pH and an overload of sodium inside the cell. During reperfusion, the sodium-proton exchanger (NHE) removes protons from the cell in an effort to restore normal pH, in turn importing sodium ions. Many strategies have been attempted to prevent reperfusion injury, including inhibition of the NHE, with little clinical effect. Using a mathematical model that we developed to study ischemia and reperfusion in cardiac cells, we found that NHE inhibition produces more severe sodium overload, largely due to adverse consequences of the delayed pH recovery produced by NHE inhibition. These results suggest that NHE inhibition alone may not be a viable strategy, and that therapies which prolong intracellular acidosis may be problematic.