Clinical Validation of a Machine-Learned, Point-of-Care System to IDENTIFY Functionally Significant Coronary Artery Disease.

Many clinical studies have shown wide performance variation in tests to identify coronary artery disease (CAD). Coronary computed tomography angiography (CCTA) has been identified as an effective rule-out test but is not widely available in the USA, particularly so in rural areas. Patients in rural areas are underserved in the healthcare system as compared to urban areas, rendering it a priority population to target with highly accessible diagnostics. We previously developed a machine-learned algorithm to identify the presence of CAD (defined by functional significance) in patients with symptoms without the use of radiation or stress. The algorithm requires 215 s temporally synchronized photoplethysmographic and orthogonal voltage gradient signals acquired at rest. The purpose of the present work is to validate the performance of the algorithm in a frozen state (i.e., no retraining) in a large, blinded dataset from the IDENTIFY trial. IDENTIFY is a multicenter, selectively blinded, non-randomized, prospective, repository study to acquire signals with paired metadata from subjects with symptoms indicative of CAD within seven days prior to either left heart catheterization or CCTA. The algorithm's sensitivity and specificity were validated using a set of unseen patient signals (n = 1816). Pre-specified endpoints were chosen to demonstrate a rule-out performance comparable to CCTA. The ROC-AUC in the validation set was 0.80 (95% CI: 0.78-0.82). This performance was maintained in both male and female subgroups. At the pre-specified cut point, the sensitivity was 0.85 (95% CI: 0.82-0.88), and the specificity was 0.58 (95% CI: 0.54-0.62), passing the pre-specified endpoints. Assuming a 4% disease prevalence, the NPV was 0.99. Algorithm performance is comparable to tertiary center testing using CCTA. Selection of a suitable cut-point results in the same sensitivity and specificity performance in females as in males. Therefore, a medical device embedding this algorithm may address an unmet need for a non-invasive, front-line point-of-care test for CAD (without any radiation or stress), thus offering significant benefits to the patient, physician, and healthcare system.

Pulmonary Hypertension Detection Non-Invasively at Point-of-Care Using a Machine-Learned Algorithm.

Artificial intelligence, particularly machine learning, has gained prominence in medical research due to its potential to develop non-invasive diagnostics. Pulmonary hypertension presents a diagnostic challenge due to its heterogeneous nature and similarity in symptoms to other cardiovascular conditions. Here, we describe the development of a supervised machine learning model using non-invasive signals (orthogonal voltage gradient and photoplethysmographic) and a hand-crafted library of 3298 features. The developed model achieved a sensitivity of 87% and a specificity of 83%, with an overall Area Under the Receiver Operator Characteristic Curve (AUC-ROC) of 0.93. Subgroup analysis showed consistent performance across genders, age groups and classes of PH. Feature importance analysis revealed changes in metrics that measure conduction, repolarization and respiration as significant contributors to the model. The model demonstrates promising performance in identifying pulmonary hypertension, offering potential for early detection and intervention when embedded in a point-of-care diagnostic system.

Development of a Non-Invasive Machine-Learned Point-of-Care Rule-Out Test for Coronary Artery Disease.

The current standard of care for coronary artery disease (CAD) requires an intake of radioactive or contrast enhancement dyes, radiation exposure, and stress and may take days to weeks for referral to gold-standard cardiac catheterization. The CAD diagnostic pathway would greatly benefit from a test to assess for CAD that enables the physician to rule it out at the point of care, thereby enabling the exploration of other diagnoses more rapidly. We sought to develop a test using machine learning to assess for CAD with a rule-out profile, using an easy-to-acquire signal (without stress/radiation) at the point of care. Given the historic disparate outcomes between sexes and urban/rural geographies in cardiology, we targeted equal performance across sexes in a geographically accessible test. Noninvasive photoplethysmogram and orthogonal voltage gradient signals were simultaneously acquired in a representative clinical population of subjects before invasive catheterization for those with CAD (gold-standard for the confirmation of CAD) and coronary computed tomographic angiography for those without CAD (excellent negative predictive value). Features were measured from the signal and used in machine learning to predict CAD status. The machine-learned algorithm achieved a sensitivity of 90% and specificity of 59%. The rule-out profile was maintained across both sexes, as well as all other relevant subgroups. A test to assess for CAD using machine learning on a noninvasive signal has been successfully developed, showing high performance and rule-out ability. Confirmation of the performance on a large clinical, blinded, enrollment-gated dataset is required before implementation of the test in clinical practice.

Effects of genetic transfection on calcium cycling pathways mediated by double-stranded adeno-associated virus in postinfarction remodeling.

OBJECTIVE: Restoring calcium sensor protein (S100A1) activity in failing hearts poses a promising therapeutic strategy. We hypothesize that cardiac overexpression of the S100A1 gene mediated by a double-stranded adeno-associated virus (scAAV) results in better functional and molecular improvements compared with the single-stranded virus (ssAAV). METHODS: Heart failure was induced by coronary artery ligation. Then, intramyocardial injections of saline, AAV9 empty capsid, scAAV9.S100A1, and ssAAV9.S100A1 were performed. Ten weeks postinfarction, all rats received cardiac evaluation; serum and tissue were collected for genetic analysis, cytokine profiling, and assessments of mitochondrial function and structure. RESULTS: Overexpression of AAV9.S100A1 improved systolic and diastolic function. Compared with control, ejection fraction was greater in treated groups (54.8% vs 32.3%, P < .05). Similarly, end-diastolic volume index was significantly less in the treated group than in control (1.14 vs 1.59 mL/cm2), whereas fractional shortening was greater in treated groups than control (26% vs 38%, P < .05). Interestingly, cardiac mechanics were significantly better when treated with double-stranded virus compared with single-stranded. Quantitative polymerase chain reaction demonstrated robust transfection of myocardium with the S100A1 gene, with more infection in the self-complimentary group compared with the single-stranded group (5.68 ± 0.44 vs 4.09 ± 0.25 log10 genome copies per 100 ng of DNA; P < .0001). Concentrations of the inflammatory cytokines were elevated in the ssAAV9/S100A1 group compared with the scAAV9/S100A1. Assessment of mitochondrial respiration and morphology demonstrated that injection of self-complementary vector saved both mitochondrial structure and function. CONCLUSIONS: Gene therapy of S100A1 can prevent pathologic postmyocardial infarction remodeling and decrease inflammatory response in ischemic heart failure.

Surgical and physiological challenges in the development of left and right heart failure in rat models.

Rodent surgical animal models of heart failure (HF) are critically important for understanding the proof of principle of the cellular alterations underlying the development of the disease as well as evaluating therapeutics. Robust, reproducible rodent models are a prerequisite to the development of pharmacological and molecular strategies for the treatment of HF in patients. Due to the absence of standardized guidelines regarding surgical technique and clear criteria for HF progression in rats, objectivity is compromised. Scientific publications in rats rarely fully disclose the actual surgical details, and technical and physiological challenges. This lack of reporting is one of the main reasons that the outcomes specified in similar studies are highly variable and associated with unnecessary loss of animals, compromising scientific assessment. This review details rat circulatory and coronary arteries anatomy, the surgical details of rat models that recreate the HF phenotype of myocardial infarction, ischemia/reperfusion, left and right ventricular pressure, and volume overload states, and summarizes the technical and physiological challenges of creating HF. The purpose of this article is to help investigators understand the underlying issues of current HF models in order to reduce variable results and ensure successful, reproducible models of HF.

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats.

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

Targeted Gene Delivery through the Respiratory System: Rationale for Intratracheal Gene Transfer.

Advances in DNA- and RNA-based technologies have made gene therapy suitable for many lung diseases, especially those that are hereditary. The main objective of gene therapy is to deliver an adequate amount of gene construct to the intended target cell, achieve stable transduction in target cells, and to produce a clinically therapeutic effect. This review focuses on the cellular organization in the normal lung and how gene therapy targets the specific cell types that are affected by pulmonary disorders caused by genetic mutations. Furthermore, it examines the pulmonary barriers that can compromise the absorption and transduction of viral vectors and genetic agents by the lung. Finally, it discusses the advantages and limitations of direct intra-tracheal gene delivery with different viral vectors in small and large animal models and in clinical trials.

Technique of Complete Heart Isolation with Continuous Cardiac Perfusion During Cardiopulmonary Bypass: New Opportunities for Gene Therapy.

Cardiopulmonary bypass (CPB) featuring complete heart isolation and continuous cardiac perfusion is a very promising approach for solving the problem of efficient gene delivery. In the technique presented here, separate pumps are used for the systemic and cardiac circuits. This system permits continuous isolated arrested heart perfusion through optimizing a number of delivery parameters including temperature, flow rate, driving pressure, ionic composition, and exposure time to the cardiac vessels. During complete cardiac isolation, the blood vector concentration trended from 11.51 ± 1.73 log genome copies (GCs)/cm3 to 9.84 ± 1.65 log GC/cm3 (p > .05). Despite restructuring a very high concentration to the heart, GCs were detectable in the systemic circuit. These values over time were near negligible by comparison but detectable 1.66 ± .26 during 20 minutes of recirculation and did not change (p > .05). After the completion of the recirculation interval and subsequent washing procedure, the initial systemic blood vector GC concentration slightly increased to 2.08 ± .38 log GCs/cm3 (p > .05). During the recirculation period, we supported flow via the cardiac circuit around 300 mL/min. In this technique of heart isolation with continuous cardiac perfusion, >99% of the vector remains in coronary circulation during recirculation period. The animal's non recirculation blood, or that in the system, was routinely tested during and after recirculation to contain much less than 1% of the original dose obtained via logging concentration of therapeutic over time. All of the sheep in this group recovered from anesthesia and received critical postoperative care, including all organ function, in the first 24-36 hours. Twenty-one sheep (84%) survived to euthanasia at 12 weeks. Average CPB time was 107 ± 19.0 minutes and cross-clamp time was 49 ± 7.9 minutes. This technology readily provides multiple pass recirculation of genes through the heart with minimal side effects of collateral expression of other organs.

Scar Size and Other Parameters for Tracking Left Ventricular Dysfunction after Induction of Myocardial Infarcts in Sheep (Ovisaries).

In humans, cardiovascular disease (CVD) is the most frequent cause of death worldwide. Myocardial infarction (MI) is a leading cause of heart failure due to myocardial impairment, yet the progression of the resultant dysfunction is often undetected after incidental or induced myocardial infarction. In this study we tracked the progression of left-sided heart failure in 6-mo-old male castrated sheep in which we created 2 models of myocardial infarction, small and large. Myocardial infarction was induced through ligation of a single branch (obtuse marginal [OM] 1) of the left circumflex coronary artery to create small (mild) infarcts and of 2 branches (OM1 and OM2) for large (severe) infarcts. Progression of heart failure was evaluated by assessing scar size, the left ventricular ejection fraction, hematology, cardiac serum biochemical biomarkers, ST elevation, and clinical observation. All parameters were assessed at baseline and at 3 wk and 3 mo after infarction, except that clinical observation of the animals was conducted daily. The different parameters differed in their usefulness: some verified appropriate creation of the model, whereas others enabled assessment of the progression of heart disease. We hypothesize that myocardial scar size, as a function of induced ischemia, coupled with left ventricular ejection fraction are predictive indicators of postinfarction cardiac dysfunction.

[GENE THERAPY POTENTIAL AS A TREATMENT FOR HEART FAILURE].

INTRODUCTION: Advances in understanding the molecular biology of heart failure, the evolution of vector technology, as well as defining the targets for therapeutic interventions has placed heart failure within the reach of gene-based therapy. During the last decade the concept of delivering cDNA encoding a therapeutic gene to failing cardiomyocytes has moved from hypothesis to the bench of preclinical applications and clinical trials. However, despite significant promise, several obstacles exist, which are described in this review. We anticipate that advances in the field will improve gene therapy in heart failure in future clinical approaches.

Use of Adeno-Associated Virus Vector for Cardiac Gene Delivery in Large-Animal Surgical Models of Heart Failure.

The advancement of gene therapy-based approaches to treat heart disease represents a need for clinically relevant animal models with characteristics equivalent to human pathologies. Rodent models of cardiac disease do not precisely reproduce heart failure phenotype and molecular defects. This has motivated researchers to use large animals whose heart size and physiological processes more similar and comparable to those of humans. Today, adeno-associated viruses (AAV)-based vectors are undoubtedly among the most promising DNA delivery vehicles. Here, AAV biology and technology are reviewed and discussed in the context of their use and efficacy for cardiac gene delivery in large-animal models of heart failure, using different surgical approaches. The remaining challenges and opportunities for the use of AAV-based vector delivery for gene therapy applications in the clinic are also highlighted.

In Situ Heart Isolation Featuring Closed Loop Recirculation: The Gold Standard for Optimum Cardiac Gene Transfer?

The concept of delivering nucleic material encoding a therapeutic gene to the heart has arduously moved from hypothesis to a variety of high potential clinical applications. Despite the promise however, the results achieved have yet to be realized due to several problems that persist in the clinic. One of these identified problems is the need for an efficient delivery method which facilitates complete cardiotropism and minimizes collateral effects. Additional parameters impacting gene delivery that most need to be improved have been identified as follows: (1) Increasing the contact time of vector in coronary circulation permitting transfer, (2) Sustained intravascular flow rate and perfusion pressure to facilitate proper kinetics, (3) Modulation of cellular permeability to increase uptake efficiency, and once in the cells (4) Enhancing transcription and translation within the transfected cardiac cells, and (5) Obtaining the global gene distribution for maximum efficacy. Recently it was hypothesized that use of cardiopulmonary bypass may facilitate cardiac-selective gene transfer and permit vector delivery in the arrested heart in isolated "closed loop" recirculating model. This system was named molecular cardiac surgery with recirculating delivery (MCARD). The key components of this approach include: isolation of the heart from systemic organs, multiple pass recirculation of vector through the coronary vasculature, and removing the residual vector from the coronary circulation to minimize collateral expression. These attributes unique to a surgical approach such as MCARD can effectively increase vector transduction efficiency in coronary vasculature.

A Needleless Liquid Jet Injection Delivery Approach for Cardiac Gene Therapy.

Fundamentally, cardiac gene therapy clinical trials have demonstrated that route efficiency is paramount in achieving maximum myocardial expression within safety limits. Gene transfer phenomena are largely influenced by physical transport principles (i.e., pressure, residence time, dispersion trafficking, mechanical resistance) that are independent of therapeutic characteristics. An alternative to intracoronary infusion methods, in an effort to improve efficiency in terms of cardiac specificity, is direct myocardial delivery via surgical injection. Direct injection methods circumvent the blood's immunological components and the cardiac system's native anatomical barriers by directly administering product into the myocardium. In addition, this approach offers the advantage of precise site selection. Two unresolved problems with direct delivery wherein the novel needleless liquid jet approach may resolve are: (1) initial therapeutic retention and (2) subsequent host responses associated with highly focal expression.In this protocol, we present a novel approach to improve direct cardiac gene delivery using a needleless liquid jet methodology. The liquid jet application is essentially a device concept that accelerates and disperses the therapeutic at a targeted myocardial site. The core hypothesis offered is that this approach, with optimized settings, could result in increased therapeutic retention in the initial delivery phase. This would theoretically result in more total myocardial expression per dose while at the same time providing a more homogenous profile around the injection site. Therefore, this would increase efficiency in terms of transduced muscle per delivery site and offer a significant improvement to standard intramuscular injection.

Molecular Cardiac Surgery with Recirculating Delivery (MCARD): Procedure and Vector Transfer.

Despite progress in clinical treatment, cardiovascular diseases are still the leading cause of morbidity and mortality worldwide. Therefore, novel therapeutic approaches are needed, targeting the underlying molecular mechanisms of disease with improved outcomes for patients. Gene therapy is one of the most promising fields for the development of new treatments for the advanced stages of cardiovascular diseases. The establishment of clinically relevant methods of gene transfer remains one of the principal limitations on the effectiveness of gene therapy. Recently, there have been significant advances in direct and transvascular gene delivery methods. The ideal gene transfer method should be explored in clinically relevant large animal models of heart disease to evaluate the roles of specific molecular pathways in disease pathogenesis. Characteristics of the optimal technique for gene delivery include low morbidity, an increased myocardial transcapillary gradient, esxtended vector residence time in the myocytes, and the exclusion of residual vector from the systemic circulation after delivery to minimize collateral expression and immune response. Here we describe myocardial gene transfer techniques with molecular cardiac surgery with recirculating delivery in a large animal model of post ischemic heart failure.

Modified mRNA as a therapeutic tool to induce cardiac regeneration in ischemic heart disease.

Ischemic heart disease (IHD) is a leading cause of morbidity and mortality in developed countries. Current pharmacological and interventional therapies provide significant improvement in the life quality of patient; however, they are mostly symptom-oriented and not curative. A high disease and economic burden of IHD requires the search for new therapeutic strategies to significantly improve patients' prognosis and quality of life. One of the main challenges during IHD is the massive loss of cardiomyocytes that possess minimal regenerative capacity. Recent understanding of the pathophysiological mechanisms underlying IHD, as well as new therapeutic approaches provide new hope for patients suffering from IHD. Synthetic modified mRNA (modRNA) is a new gene delivery vector that is increasingly used in in vivo applications. modRNA is a relatively stable, non-immunogenic, highly-expressed molecule that has been shown to mediate high and transient expression of proteins in different type of cells and tissues including cardiomyocytes. modRNA properties, together with its expression kinetics in the heart make it an attractive option for the treatment of IHD, especially after myocardial infarction. In this review we discuss the role of gene therapy in cardiac regeneration as an approach to treat IHD; traditional and innovative gene delivery methods; and focus specifically on modRNA structure, mode of delivery, and its use for the induction of endogenous regenerative capacity, mainly in the context of IHD. WIREs Syst Biol Med 2017, 9:e1367. doi: 10.1002/wsbm.1367 For further resources related to this article, please visit the WIREs website.

Gene Therapy in Heart Failure.

Heart failure is a significant burden to the global healthcare system and represents an underserved market for new pharmacologic strategies, especially therapies which can address root cause myocyte dysfunction. Modern drugs, surgeries, and state-of-the-art interventions are costly and do not improve survival outcome measures. Gene therapy is an attractive strategy, whereby selected gene targets and their associated regulatory mechanisms can be permanently managed therapeutically in a single treatment. This in theory could be sustainable for the patient's life. Despite the promise, however, gene therapy has numerous challenges that must be addressed together as a treatment plan comprising these key elements: myocyte physiologic target validation, gene target manipulation strategy, vector selection for the correct level of manipulation, and carefully utilizing an efficient delivery route that can be implemented in the clinic to efficiently transfer the therapy within safety limits. This chapter summarizes the key developments in cardiac gene therapy from the perspective of understanding each of these components of the treatment plan. The latest pharmacologic gene targets, gene therapy vectors, delivery routes, and strategies are reviewed.

Mitigation of myocardial fibrosis by molecular cardiac surgery-mediated gene overexpression.

OBJECTIVE: Heart failure is accompanied by up-regulation of transforming growth factor beta signaling, accumulation of collagen and dysregulation of sarcoplasmic reticulum calcium adenosine triphosphatase cardiac isoform 2a (SERCA2a). We examined the fibrotic response in small and large myocardial infarct, and the effect of overexpression of the SERCA2a gene. METHODS: Ischemic cardiomyopathy was induced via creation of large or small infarct in 26 sheep. Animals were divided into 4 groups: small infarct; large infarct with heart failure; gene-treated (large infarct with heart failure followed by adeno-associated viral vector, serotype 1.SERCA2a gene construct transfer by molecular cardiac surgery with recirculating delivery); and control. RESULTS: Heart failure was significantly less pronounced in the gene-treated and small-infarct groups than in the large-infarct group. Expression of transforming growth factor beta signaling components was significantly higher in the large-infarct group, compared with the small-infarct and gene-treated groups. Both the angiotensin II type 1 receptor and angiotensin II were significantly elevated in the small- and large-infarct groups, whereas gene treatment diminished this effect. Active fibrosis with de novo collagen synthesis was evident in the large-infarct group; the small-infarct and gene-treated groups showed less fibrosis, with a lower ratio of de novo to mature collagen. CONCLUSIONS: The data presented provide evidence that progression of fibrosis is mediated through increased transforming growth factor beta and angiotensin II signaling, which is mitigated by increased SERCA2a gene expression.

Gene Therapy in Cardiac Surgery: Clinical Trials, Challenges, and Perspectives.

The concept of gene therapy was introduced in the 1970s after the development of recombinant DNA technology. Despite the initial great expectations, this field experienced early setbacks. Recent years have seen a revival of clinical programs of gene therapy in different fields of medicine. There are many promising targets for genetic therapy as an adjunct to cardiac surgery. The first positive long-term results were published for adenoviral administration of vascular endothelial growth factor with coronary artery bypass grafting. In this review we analyze the past, present, and future of gene therapy in cardiac surgery. The articles discussed were collected through PubMed and from author experience. The clinical trials referenced were found through the Wiley clinical trial database (http://www.wiley.com/legacy/wileychi/genmed/clinical/) as well as the National Institutes of Health clinical trial database (Clinicaltrials.gov).

The role of microRNAs in cardiac development and regenerative capacity.

The mammalian heart has long been considered to be a postmitotic organ. It was thought that, in the postnatal period, the heart underwent a transition from hyperplasic growth (more cells) to hypertrophic growth (larger cells) due to the conversion of cardiomyocytes from a proliferative state to one of terminal differentiation. This hypothesis was gradually disproven, as data were published showing that the myocardium is a more dynamic tissue in which cardiomyocyte karyokinesis and cytokinesis produce new cells, leading to the hyperplasic regeneration of some of the muscle mass lost in various pathological processes. microRNAs have been shown to be critical regulators of cardiomyocyte differentiation and proliferation and may offer the novel opportunity of regenerative hyperplasic therapy. Here we summarize the relevant processes and recent progress regarding the functions of specific microRNAs in cardiac development and regeneration.