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An ensemble of in vitro cardiac tissue models has been developed over the past several decades to aid our understanding of complex cardiovascular disorders using a reductionist approach. These approaches often rely on recapitulating single or multiple clinically relevant end points in a dish indicative of the cardiac pathophysiology. The possibility to generate disease-relevant and patient-specific human induced pluripotent stem cells has further leveraged the utility of the cardiac models as screening tools at a large scale. To elucidate biological mechanisms in the cardiac models, it is critical to integrate physiological cues in form of biochemical, biophysical, and electromechanical stimuli to achieve desired tissue-like maturity for a robust phenotyping. Here, we review the latest advances in the directed stem cell differentiation approaches to derive a wide gamut of cardiovascular cell types, to allow customization in cardiac model systems, and to study diseased states in multiple cell types. We also highlight the recent progress in the development of several cardiovascular models, such as cardiac organoids, microtissues, engineered heart tissues, and microphysiological systems. We further expand our discussion on defining the context of use for the selection of currently available cardiac tissue models. Last, we discuss the limitations and challenges with the current state-of-the-art cardiac models and highlight future directions.

Rationale: The BMPs (bone morphogenetic proteins) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular diseases, such as arteriovenous malformations. Objective: A computational model predicted that BMP4 and BMP9 and their inhibitors MGP (matrix gamma-carboxyglutamic acid [Gla] protein) and CV2 (crossveinless-2) would form a regulatory system consisting of negative feedback loops with time delays and that BMP9 would trigger oscillatory expression of the 2 inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth. Methods and Results: Oscillations in the expression of MGP and CV2 were detected in endothelial cells in vitro, using quantitative real-time polymerase chain reaction and immunoblotting. These organized temporally downstream BMP-related activities, including expression of stalk-cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing. Conclusions: Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilize the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.

A continuous supply of oxygen is essential for the survival of multi-cellular organisms. The understanding of how this supply is regulated in the microvasculature has evolved from viewing erythrocytes (red blood cells, RBCs) as passive carriers of oxygen to recognizing the complex interplay between hemoglobin and oxygen, carbon dioxide, and nitric oxide – the three-gas respiratory cycle – that insures adequate oxygen and nutrient delivery to meet local metabolic demand. In this context, it is blood flow not blood oxygen content that is the main driver of tissue oxygenation by RBCs. Herein we review the lines of experimentation that led to this understanding of RBC function; from the foundational understanding of allosteric regulation of oxygen binding in hemoglobin in the stereochemical model of Perutz, to blood flow autoregulation (hypoxic vasodilation governing oxygen delivery) observed by Guyton, to current understanding that centers on S-nitrosylation of hemoglobin (aka S-nitrosohemoglobin; SNO-Hb) as a purveyor of oxygen-dependent vasodilatory activity. Notably, hypoxic vasodilation is recapitulated by native SNO-replete RBCs and by SNO-Hb itself, whereby SNO is released from hemoglobin and RBCs during deoxygenation, in proportion to the degree of hemoglobin deoxygenation, to regulate vessels directly. In addition, we discuss how dysregulation of this system through genetic mutation in hemoglobin or through disease is a common factor in oxygenation pathologies resulting from microcirculatory impairment, including sickle cell disease, ischemic heart disease and heart failure. We then conclude by identifying potential therapeutic interventions to correct deficits in RBC-mediated vasodilation to improve oxygen delivery—steps towards effective microvasculature-targeted therapies. To the extent that diseases of the heart, lungs, and blood are associated with impaired tissue oxygenation, the development of new therapies based on the three-gas respiratory system have the potential to improve the well-being of millions of patients.

Machine learning (ML), a branch of artificial intelligence, where machines learn from big data, is at the crest of a technological wave of change sweeping society. Cardiovascular medicine is at the forefront of many ML applications, and there is a significant effort to bring them into mainstream clinical practice. In the field of cardiac electrophysiology, ML applications have also seen a rapid growth and popularity, particularly the use of ML in the automatic interpretation of ECGs, which has been extensively covered in the literature. Much lesser known are the other aspects of ML application in cardiac electrophysiology and arrhythmias, such as those in basic science research on arrhythmia mechanisms, both experimental and computational; in the development of better techniques for mapping of cardiac electrical function; and in translational research related to arrhythmia management. In the current review, we examine comprehensively such ML applications as they match the scope of this journal. The current review is organized in 3 parts. The first provides an overview of general ML principles and methodologies that will afford readers of the necessary information on the subject, serving as the foundation for inviting further ML applications in arrhythmia research. The basic information we provide can serve as a guide on how one might design and conduct an ML study. The second part is a review of arrhythmia and electrophysiology studies in which ML has been utilized, highlighting the broad potential of ML approaches. For each subject, we outline comprehensively the general topics, while reviewing some of the research advances utilizing ML under the subject. Finally, we discuss the main challenges and the perspectives for ML-driven cardiac electrophysiology and arrhythmia research.

RATIONALE: Fibroblast growth factor homologous factors (FHFs) are key regulators of sodium channel inactivation. Mutations in these critical proteins have been implicated in human diseases including Brugada syndrome, idiopathic ventricular arrhythmias, and epileptic encephalopathy. The underlying ionic mechanisms by which reduced sodium channel availability in Fhf2 knockout mice predisposes to abnormal excitability at the tissue level are not well defined. OBJECTIVE: Using animal models and theoretical multicellular linear strands, we examined how FHF2 orchestrates the interdependency of sodium, calcium, and gap junctional conductances to safeguard cardiac conduction. METHODS AND RESULTS: Fhf2(KO) mice were challenged by reducing calcium conductance using verapamil or by reducing gap junctional conductance using carbenoxolone or by backcrossing into a connexin 43 heterozygous (Cx43(+/−)) background. All conditions produced conduction block in Fhf2(KO) mice, with Fhf2(WT) showing normal impulse propagation. To explore the ionic mechanisms of block in Fhf2(KO) hearts, multicellular linear strand models incorporating FHF2-deficient sodium channel inactivation properties were constructed and faithfully recapitulated conduction abnormalities seen in mutant hearts. The mechanisms of conduction block in mutant strands with reduced calcium conductance or gap junction uncoupling are very different. Enhanced sodium channel inactivation due to FHF2 deficiency shifts dependence onto calcium current to sustain electrotonic driving force, axial current flow, and action potential generation from cell-to-cell. In the setting of gap junction uncoupling, slower charging time from upstream cells conspires with accelerated sodium channel inactivation in mutant strands to prevent sufficient downstream cell charging for action potential propagation. CONCLUSIONS: FHF2-dependent effects on sodium channel inactivation ensure adequate sodium current reserve to safeguard against numerous threats to reliable cardiac impulse propagation.

Systems medicine is a holistic approach to deciphering the complexity of human physiology in health and disease. In essence, a living body is constituted of networks of dynamically interacting units (molecules, cells, organs, etc) that underlie its collective functions. Declining resilience because of aging and other chronic environmental exposures drives the system to transition from a health state to a disease state; these transitions, triggered by acute perturbations or chronic disturbance, manifest as qualitative shifts in the interactions and dynamics of the disease-perturbed networks. Understanding health-to-disease transitions poses a high-dimensional nonlinear reconstruction problem that requires deep understanding of biology and innovation in study design, technology, and data analysis. With a focus on the principles of systems medicine, this Review discusses approaches for deciphering this biological complexity from a novel perspective, namely, understanding how disease-perturbed networks function; their study provides insights into fundamental disease mechanisms. The immediate goals for systems medicine are to identify early transitions to cardiovascular (and other chronic) diseases and to accelerate the translation of new preventive, diagnostic, or therapeutic targets into clinical practice, a critical step in the development of personalized, predictive, preventive, and participatory (P4) medicine.

Despite sophisticated and increasingly effective assaults on traditional atherosclerotic risk factors, in particular LDL (low-density lipoprotein cholesterol) and most recently inflammation, there remains a large, unmet coronary heart disease (CHD) burden.1 Modern experimental and therapeutic programs have focused on a small number of biological pathways, so it is possible that we have reached the point where to identify additional causal mechanisms and create novel therapies, fundamentally new approaches are required. The recently developed One Brave Idea program, generously sponsored by the American Heart Association and Verily with AstraZeneca, was designed to identify alternative strategies to address CHD. In this perspective, we examine the existing frameworks in atherosclerosis biology, highlighting their successes and focusing on areas where there may be missing information. We will outline general principles which would enable biomedical researchers to identify and capture relevant missing information in CHD. Central to the program are efforts to radically improve the phenotypic repertoire of human biology and to define environmental drivers of disease, including nutrition, at a resolution and scale only feasible in the current connected era. This approach also implies the potential for a universal objective framework for the definition of personal health or disease, harmonized across the entire biomedical community to accelerate the integration of discovery more fully with prevention and clinical care. To find disease causation, one must first detect exposures (environmental or genetic) of large enough effect size that they can be clearly associated with specific outcomes. Many associations may be observed, but only a small number of such correlations are truly causal or represent targets for therapeutic intervention. Defining causality or therapeutic efficacy is demanding, particularly when observations are sparse and temporally distant from the outcomes of relevance. By measuring both exposures and outcomes at a more granular level and across time, causality is more …

The rampant misuse of the P value and of its stated meaning lead the American Statistical Association to comment, researchers often wish to turn a P value into a statement about the truth of a null hypothesis, or about the probability that random chance produced the observed data. The P value is neither. It is a statement about data in relation to a specified hypothetical explanation and is not a statement about the explanation itself.1 The American Statistical Association is not alone in its concern: a host of recent literature provides context for the desire to use statistical inference to reinforce rigor and reproducibility in scientific research.2–4 A central focus of this literature is the widely acknowledged and severe limitations we impose on ourselves with a blind and naive adherence to the exclusive use of P values for understanding significance of research findings. Counterpoint, see p 1046 Response by Moye and Cohen, see p 1051 The inadequacy of P values as a singular and standard criteria for significance in the analysis of clinical trials has been known and communicated by statisticians for >35 years.5 Given the limitations of significance testing, can we simply, as suggested by Moye and Cohen, resolve to walk away from P values? Many have argued the use of methods that emphasize estimation over testing, such as confidence, credibility, or prediction intervals that can be used in place of or in addition to P values.6–8 Others have lobbied for alternative measures of evidence, such as likelihood ratios or Bayesian methodologies, which leverage a priori information about the probability of different magnitudes of effect that result from the treatment and modify the estimation of the effects based on the observed data.9–11 Still, others propose approaches such as …

The mTOR (mechanistic target of rapamycin) is a master regulator of several crucial cellular processes, including protein synthesis, cellular growth, proliferation, autophagy, lysosomal function, and cell metabolism. mTOR interacts with specific adaptor proteins to form 2 multiprotein complexes, called mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2). In the cardiovascular system, the mTOR pathway regulates both physiological and pathological processes in the heart. It is needed for embryonic cardiovascular development and for maintaining cardiac homeostasis in postnatal life. Studies involving mTOR loss-of-function models revealed that mTORC1 activation is indispensable for the development of adaptive cardiac hypertrophy in response to mechanical overload. mTORC2 is also required for normal cardiac physiology and ensures cardiomyocyte survival in response to pressure overload. However, partial genetic or pharmacological inhibition of mTORC1 reduces cardiac remodeling and heart failure in response to pressure overload and chronic myocardial infarction. In addition, mTORC1 blockade reduces cardiac derangements induced by genetic and metabolic disorders and has been reported to extend life span in mice. These studies suggest that pharmacological targeting of mTOR may represent a therapeutic strategy to confer cardioprotection, although clinical evidence in support of this notion is still scarce. This review summarizes and discusses the new evidence on the pathophysiological role of mTOR signaling in the cardiovascular system.

Lower extremity peripheral artery disease (PAD) is frequently underdiagnosed, in part because of the wide variety of leg symptoms manifested by patients with PAD and in part because of the high prevalence of asymptomatic PAD. In primary care medical practices, 30% to 60% of patients with PAD report no exertional leg symptoms and ≈45% to 50% report exertional leg symptoms that are not consistent with classic intermittent claudication. The prevalence and extent of functional impairment and functional decline in PAD may also be underappreciated. Functional impairment and functional decline are common in PAD, even among those who are asymptomatic. Lower extremity ischemia is also associated with pathophysiologic changes in calf skeletal muscle, including smaller calf muscle area, increased calf muscle fat content, impaired leg strength, and impaired metabolic function. People with severe PAD have poorer peroneal nerve conduction velocity compared with people with mild PAD or no PAD. The degree of ischemia-related pathophysiologic changes in lower extremity muscles and peripheral nerves of people with PAD are associated with the degree of functional impairment. New interventions are needed to improve functional performance and prevent mobility loss in the large number of patients with PAD, including in those who are asymptomatic or who have exertional leg symptoms other than claudication.

Elevated levels of inflammatory mediators have been identified in patients with heart failure, including heart failure with reduced and preserved ejection fraction, as well as acute decompensated heart failure. Moreover, experimental studies have shown repeatedly that activation of inflammation in the heart provokes left ventricular remodeling and left ventricular dysfunction. Nonetheless, phase III clinical trials that have attempted to antagonize inflammatory mediators have been negative with respect to the primary end points of the trials, and in some patients, resulted in worsening heart failure or death. The following review will discuss how recent developments in the field of innate immunity have advanced our understanding of the role of inflammation in the pathogenesis of heart failure and will discuss the negative outcomes of the existing clinical trials in light of this new information.

Excess weight gain, especially when associated with increased visceral adiposity, is a major cause of hypertension, accounting for 65% to 75% of the risk for human primary (essential) hypertension. Increased renal tubular sodium reabsorption impairs pressure natriuresis and plays an important role in initiating obesity hypertension. The mediators of abnormal kidney function and increased blood pressure during development of obesity hypertension include (1) physical compression of the kidneys by fat in and around the kidneys, (2) activation of the renin–angiotensin–aldosterone system, and (3) increased sympathetic nervous system activity. Activation of the renin–angiotensin–aldosterone system is likely due, in part, to renal compression, as well as sympathetic nervous system activation. However, obesity also causes mineralocorticoid receptor activation independent of aldosterone or angiotensin II. The mechanisms for sympathetic nervous system activation in obesity have not been fully elucidated but may require leptin and activation of the brain melanocortin system. With prolonged obesity and development of target organ injury, especially renal injury, obesity-associated hypertension becomes more difficult to control, often requiring multiple antihypertensive drugs and treatment of other risk factors, including dyslipidemia, insulin resistance and diabetes mellitus, and inflammation. Unless effective antiobesity drugs are developed, the effect of obesity on hypertension and related cardiovascular, renal and metabolic disorders is likely to become even more important in the future as the prevalence of obesity continues to increase.

Diet, exercise, stress, and sleep are receiving attention as environmental modifiers of chronic inflammatory diseases, including atherosclerosis, the culprit condition of myocardial infarction and stroke. Accumulating data indicate that psychosocial stress and a high-fat, high-cholesterol diet aggravate cardiovascular disease, whereas regular physical activity and healthy sleeping habits help prevent it. Here, we raise the possibility that inflammation-associated leukocyte production plays a causal role in lifestyle effects on atherosclerosis progression. Specifically, we explore whether and how potent real-life disease modifiers influence hematopoiesis’ molecular and cellular machinery. Lifestyle, we hypothesize, may rearrange hematopoietic topography, diverting production from the bone marrow to the periphery, thus propagating a quantitative and qualitative drift of the macrophage supply chain. These changes may involve progenitor-extrinsic and intrinsic communication nodes that connect organ systems along neuroimmune and immunometabolic axes, ultimately leading to an altered number and phenotype of lesional macrophages. We propose that, in conjunction with improved public health policy, future therapeutics could aim to modulate the quantitative and qualitative output, as well as the location, of the hematopoietic tree to decrease the risk of atherosclerosis complications.

Vascular stiffness is a mechanical property of the vessel wall that affects blood pressure, permeability, and inflammation. As a result, vascular stiffness is a key driver of (chronic) human disorders, including pulmonary arterial hypertension, kidney disease, and atherosclerosis. Responses of the endothelium to stiffening involve integration of mechanical cues from various sources, including the extracellular matrix, smooth muscle cells, and the forces that derive from shear stress of blood. This response in turn affects endothelial cell contractility, which is an important property that regulates endothelial stiffness, permeability, and leukocyte–vessel wall interactions. Moreover, endothelial stiffening reduces nitric oxide production, which promotes smooth muscle cell contraction and vasoconstriction. In fact, vessel wall stiffening, and microcirculatory endothelial dysfunction, precedes hypertension and thus underlies the development of vascular disease. Here, we review the cross talk among vessel wall stiffening, endothelial contractility, and vascular disease, which is controlled by Rho-driven actomyosin contractility and cellular mechanotransduction. In addition to discussing the various inputs and relevant molecular events in the endothelium, we address which actomyosin-regulated changes at cell adhesion complexes are genetically associated with human cardiovascular disease. Finally, we discuss recent findings that broaden therapeutic options for targeting this important mechanical signaling pathway in vascular pathogenesis.

Three-dimensional (3D) printing technology allows for the translation of a 2-dimensional medical imaging study into a physical replica of a patient’s individual anatomy. 3D printed models can facilitate a deeper understanding of complex patient anatomy and can aid in presurgical decision-making.1 Although there are 3D printing case reports in almost every subspecialty of medicine to date, the rate of adoption in the field of congenital heart disease (CHD) is particularly advanced.2,3 This is due, in no small part, to the fact that the heart is a hollow organ, which makes it a perfect substrate for 3D printing. More importantly, medical decision-making in CHD is informed by assessment of the anatomic morphology of the heart because cardiac pathology is a direct manifestation of the underlying 3D structure. Reports on the application of 3D printing in the study and treatment of CHD are accumulating rapidly; these studies cover uses, including advanced visualization, surgical planning, and education.4 Individual case reports and small studies indicate the potential to improve patient outcomes using patient-specific 3D models. There is a growing body of literature that demonstrates the value of 3D models in decision-making,5 procedural planning,5–7 and postoperative care simulation.8,9 Two specific cases are illustrated for the reader’s interest in Figures 1 and 2. Figure 1. After surgical repair of a superior sinus venosus defect and anomalous pulmonary venous drainage, a 4-year-old girl was found to have a long-segment stenosis of the superior vena cava (SVC) as it entered the right atrium, anterior to the right upper pulmonary venous baffle. A 3-dimensional (3D) printed model created from the magnetic resonance imaging (MRI) data more clearly demonstrated the relationship of the SVC stenosis to the right upper pulmonary vein baffle, giving operators the confidence to proceed with …

Rationale: Viral myocarditis is a life-threatening illness that may lead to heart failure or cardiac arrhythmias. A major causative agent for viral myocarditis is the B3 strain of coxsackievirus, a positive-sense RNA enterovirus. However, human cardiac tissues are difficult to procure in sufficient enough quantities for studying the mechanisms of cardiac-specific viral infection. Objective: This study examined whether human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) could be used to model the pathogenic processes of coxsackievirus-induced viral myocarditis and to screen antiviral therapeutics for efficacy. Methods and Results: hiPSC-CMs were infected with a luciferase-expressing coxsackievirus B3 strain (CVB3-Luc). Brightfield microscopy, immunofluorescence, and calcium imaging were used to characterize virally infected hiPSC-CMs for alterations in cellular morphology and calcium handling. Viral proliferation in hiPSC-CMs was quantified using bioluminescence imaging. Antiviral compounds including interferonβ1, ribavirin, pyrrolidine dithiocarbamate, and fluoxetine were tested for their capacity to abrogate CVB3-Luc proliferation in hiPSC-CMs in vitro. The ability of these compounds to reduce CVB3-Luc proliferation in hiPSC-CMs was consistent with reported drug effects in previous studies. Mechanistic analyses via gene expression profiling of hiPSC-CMs infected with CVB3-Luc revealed an activation of viral RNA and protein clearance pathways after interferonβ1 treatment. Conclusions: This study demonstrates that hiPSC-CMs express the coxsackievirus and adenovirus receptor, are susceptible to coxsackievirus infection, and can be used to predict antiviral drug efficacy. Our results suggest that the hiPSC-CM/CVB3-Luc assay is a sensitive platform that can screen novel antiviral therapeutics for their effectiveness in a high-throughput fashion.

Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.

Impulse propagation in the heart depends on the excitability of individual cardiomyocytes, impulse transmission between adjacent myocytes, and the 3-dimensional arrangement of those cells. Here, we review the role of each of these factors in normal and aberrant cardiac electric activation, with particular emphasis on the effects of 3-dimensional myocyte architecture at the tissue scale. The analysis draws on findings from in vivo and in vitro experiments, as well as biophysically based computer models that have been used to integrate and interpret these experimental data. It indicates that discontinuous arrangement of myocytes and extracellular connective tissue at the tissue scale can give rise to current source-to-sink mismatch, spatiotemporal distribution of refractoriness, and rate-sensitive electric instability, which contribute to the initiation and maintenance of reentrant cardiac arrhythmia. This exacerbates the risk of rhythm disturbance associated with heart disease. We conclude that structure-based, multiscale computer models that incorporate accurate information about local cellular electric activity provide a powerful platform for investigating the basis of reentrant cardiac arrhythmia. However, it is important that these models capture key features of structure and related electric function at the tissue scale.

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca 2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca 2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca 2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

Rationale: Electrogram-based catheter ablation, targeting complex fractionated atrial electrograms (CFAEs), is empirically known to be effective in halting persistent/permanent atrial fibrillation (AF). However, the mechanisms underlying CFAEs and electrogram-based ablation remain unclear. Objective: Because atrial fibrosis is associated with persistent/permanent AF, we hypothesized that electrotonic interactions between atrial myocytes and fibroblasts play an important role in CFAE genesis and electrogram-based catheter ablation. Methods and Results: We used a human atrial tissue model in heart failure and simulated propagation and spiral wave reentry with and without regionally proliferated fibroblasts. Coupling of fibroblasts to atrial myocytes resulted in shorter action potential duration, slower conduction velocity, and lower excitability. Consequently, heterogeneous fibroblast proliferation in the myocardial sheet resulted in frequent spiral wave breakups, and the bipolar electrograms recorded at the fibroblast proliferation area exhibited CFAEs. The simulations demonstrated that ablation targeting such fibroblast-derived CFAEs terminated AF, resulting from the ablation site transiently pinning the spiral wave and then pushing it out of the fibroblast proliferation area. CFAEs could not be attributed to collagen accumulation alone. Conclusions: Fibroblast proliferation in atria might be responsible for the genesis of CFAEs during persistent/permanent AF. Our findings could contribute to better understanding of the mechanisms underlying CFAE-targeted AF ablation.

The goal of systems biology is to relate events at the molecular level to more integrated scales from organelle to cell, tissue, and living organism. Here, we review how normal and abnormal excitation–contraction coupling properties emerge from the protein scale, where behaviors are dominated by randomness, to the cell and tissue scales, where heart has to beat with reliable regularity for a lifetime. Beginning with the fundamental unit of excitation–contraction coupling, the couplon where L-type Ca channels in the sarcolemmal membrane adjoin ryanodine receptors in the sarcoplasmic reticulum membrane, we show how a network of couplons with 3 basic properties (random activation, refractoriness, and recruitment) produces the classic physiological properties of excitation–contraction coupling and, under pathophysiological conditions, leads to Ca alternans and Ca waves. Moving to the tissue scale, we discuss how cellular Ca alternans and Ca waves promote both reentrant and focal arrhythmias in the heart. Throughout, we emphasize the qualitatively novel properties that emerge at each new scale of integration.

Excitation–contraction coupling in the cardiac myocyte is mediated by a number of highly integrated mechanisms of intracellular Ca 2+ transport. The complexity and integrative nature of heart cell electrophysiology and Ca 2+ cycling has led to an evolution of computational models that have played a crucial role in shaping our understanding of heart function. An important emerging theme in systems biology is that the detailed nature of local signaling events, such as those that occur in the cardiac dyad, have important consequences at higher biological scales. Multiscale modeling techniques have revealed many mechanistic links between microscale events, such as Ca 2+ binding to a channel protein, and macroscale phenomena, such as excitation–contraction coupling gain. Here, we review experimentally based multiscale computational models of excitation–contraction coupling and the insights that have been gained through their application.

Abstract : Historically, milestones in science are usually associated with methodological breakthroughs. Likewise, the advent of electrocardiography, microelectrode recordings and more recently optical mapping have ushered in new periods of significance of advancement in elucidating basic mechanisms in cardiac electrophysiology. As with any novel technique, however, data interpretation is challenging and should be approached with caution, as it cannot be simply extrapolated from previously used methodologies and with experience and time eventually becomes validated. A good example of this is the use of optical mapping in the sinoatrial node (SAN): when microelectrode and optical recordings are obtained from the same site in myocardium, significantly different results may be noted with respect to signal morphology and as a result have to be interpreted by a different set of principles. Given the rapid spread of the use of optical mapping, careful evaluation must be made in terms of methodology with respect to interpretation of data gathered by optical sensors from fluorescent potential-sensitive dyes. Different interpretations of experimental data may lead to different mechanistic conclusions. This review attempts to address the origin and interpretation of the “double component” morphology in the optical action potentials obtained from the SAN region. One view is that these 2 components represent distinctive signals from the SAN and atrial cells and can be fully separated with signal processing. A second view is that the first component preceding the phase 0 activation represents the membrane currents and intracellular calcium transients induced diastolic depolarization from the SAN. Although the consensus from both groups is that ionic mechanisms, namely the joint action of the membrane and calcium automaticity, are important in the SAN function, it is unresolved whether the double-component originates from the recording methodology or represents the underlying physiology. This overview aims to advance a common understanding of the basic principles of optical mapping in complex 3D anatomic structures.

Rationale: Although connexin changes are important for the ventricular arrhythmic substrate in congestive heart failure (CHF), connexin alterations during CHF-related atrial arrhythmogenic remodeling have received limited attention. Objective: To analyze connexin changes and their potential contribution to the atrial fibrillation (AF) substrate during the development and reversal of CHF. Methods and Results: Three groups of dogs were studied: CHF induced by 2-week ventricular tachypacing (240 bpm, n=15); CHF dogs allowed a 4-week nonpaced recovery interval after 2-week tachypacing (n=16); and nonpaced sham controls (n=19). Left ventricular (LV) end-diastolic pressure and atrial refractory periods increased with CHF and normalized on CHF recovery. CHF caused abnormalities in atrial conduction indexes and increased the duration of burst pacing-induced AF (DAF, from 22±7 seconds in control to 1100±171 seconds, P P P Conclusions: CHF causes atrial connexin changes, but these are not essential for CHF-related conduction disturbances and AF promotion, which are rather related primarily to fibrotic interruption of muscle bundle continuity.

Rationale: Variability in delivery of oxygen can lead to electric instability in the myocardium and the generation of arrhythmias. In addition ischemic heart disease and angina are associated with an increase in circulating catecholamines that further increases the risk of developing ventricular tachyarrhythmias. Objective: We investigated the net effects of acute hypoxia and catecholamines on the cardiac action potential. Methods and Results: We incorporated all published data on the effects of hypoxia on the late Na + current ( I Na-L ), the fast Na + current ( I Na ), the basal L-type Ca 2+ channel current ( I Ca-L ), and the slow ( I Ks ) and rapid components of the delayed rectifier K + -current ( I Kr ) in the absence and presence of β-adrenergic receptor (β-AR) stimulation into the Luo–Rudy model of the action potential. Hypoxia alone had little effect on the action potential configuration or action potential duration. However in the presence of β-AR stimulation, hypoxia caused a prolongation of the action potential and early afterdepolarizations (EADs) and spontaneous tachycardia were induced. Experiments performed in guinea pig ventricular myocytes confirmed the modeling results. Conclusions: EADs occur predominantly because of the increased sensitivity of I Ca-L to β-AR stimulation during hypoxia. β-AR stimulation is necessary to induce EADs as EADs are never observed during hypoxia in the absence of β-AR stimulation.

The multifunctional Ca 2+ /calmodulin-dependent protein kinase II δ C (CaMKIIδ C ) is found in the macromolecular complex of type 2 ryanodine receptor (RyR2) Ca 2+ release channels in the heart. However, the functional role of CaMKII-dependent phosphorylation of RyR2 is highly controversial. To address this issue, we expressed wild-type, constitutively active, or dominant-negative CaMKIIδ C via adenoviral gene transfer in cultured adult rat ventricular myocytes. CaMKII-mediated phosphorylation of RyR2 was reduced, enhanced, or unaltered by dominant-negative, constitutively active, or wild-type CaMKIIδ C expression, whereas phosphorylation of phospholamban at Thr17, an endogenous indicator of CaMKII activity, was at 73%, 161%, or 115% of the control group expressing β-galactosidase (β-gal), respectively. In parallel with the phospholamban phosphorylation, the decay kinetics of global Ca 2+ transients was slowed, accelerated, or unchanged, whereas spontaneous Ca 2+ spark activity was hyperactive, depressed, or unchanged in dominant-negative, constitutively active, or wild-type CaMKIIδ C groups, respectively. When challenged by high extracellular Ca 2+ , both wild-type and constitutively active CaMKIIδ C protected the cells from store overload-induced Ca 2+ release, manifested by a ≈60% suppression of Ca 2+ waves (at 2 to 20 mmol/L extracellular Ca 2+ ) in spite of an elevated sarcoplasmic reticulum Ca 2+ content, whereas dominant-negative CaMKIIδ C promoted Ca 2+ wave production (at 20 mmol/L Ca 2+ ) with significantly depleted sarcoplasmic reticulum Ca 2+ . Taken together, our data support the notion that CaMKIIδ C negatively regulates RyR2 activity and spontaneous sarcoplasmic reticulum Ca 2+ release, thereby affording a negative feedback that stabilizes local and global Ca 2+ -induced Ca 2+ release in the heart.

Supplemental Digital Content is available in the text., Rationale: Repolarization alternans (RA) are associated with arrhythmogenesis. Animal studies have revealed potential mechanisms, but human-focused studies are needed. RA generation and frequency dependence may be determined by cell-to-cell variability in protein expression, which is regulated by genetic and external factors. Objective: To characterize in vivo RA in human and to investigate in silico using human models, the ionic mechanisms underlying the frequency-dependent differences in RA behavior identified in vivo. Methods and Results: In vivo electrograms were acquired at 240 sites covering the epicardium of 41 patients at 6 cycle lengths (600–350 ms). In silico investigations were conducted using a population of biophysically detailed human models incorporating variability in protein expression and calibrated using in vivo recordings. Both in silico and in vivo, 2 types of RA were identified, with Fork- and Eye-type restitution curves, based on RA persistence or disappearance, respectively, at fast pacing rates. In silico simulations show that RA are strongly correlated with fluctuations in sarcoplasmic reticulum calcium, because of strong release and weak reuptake. Large L-type calcium current conductance is responsible for RA disappearance at fast frequencies in Eye-type (30% larger in Eye-type versus Fork-type; P

The modern treatment of cardiac arrest is an increasingly complex medical procedure with a rapidly changing array of therapeutic approaches designed to restore life to victims of sudden death. The 2 primary goals of providing artificial circulation and defibrillation to halt ventricular fibrillation remain of paramount importance for saving lives. They have undergone significant improvements in technology and dissemination into the community subsequent to their establishment 60 years ago. The evolution of artificial circulation includes efforts to optimize manual cardiopulmonary resuscitation, external mechanical cardiopulmonary resuscitation devices designed to augment circulation, and may soon advance further into the rapid deployment of specially designed internal emergency cardiopulmonary bypass devices. The development of defibrillation technologies has progressed from bulky internal defibrillators paddles applied directly to the heart, to manually controlled external defibrillators, to automatic external defibrillators that can now be obtained over-the-counter for widespread use in the community or home. But the modern treatment of cardiac arrest now involves more than merely providing circulation and defibrillation. As suggested by a 3-phase model of treatment, newer approaches targeting patients who have had a more prolonged cardiac arrest include treatment of the metabolic phase of cardiac arrest with therapeutic hypothermia, agents to treat or prevent reperfusion injury, new strategies specifically focused on pulseless electric activity, which is the presenting rhythm in at least one third of cardiac arrests, and aggressive post resuscitation care. There are discoveries at the cellular and molecular level about ischemia and reperfusion pathobiology that may be translated into future new therapies. On the near horizon is the combination of advanced cardiopulmonary bypass plus a cocktail of multiple agents targeted at restoration of normal metabolism and prevention of reperfusion injury, as this holds the promise of restoring life to many patients for whom our current therapies fail.

Vascular dysfunction is causally contributing to many diseases, including but not limited to cardiovascular disease, which is still the leading cause of death in the Western world. The endothelium that lines the inner wall of the blood vessels plays a critical role in the pathobiology of these illnesses. Particularly after ischemia or injury, the growth of new blood vessels, driven by endothelial expansion, is essential to maintain oxygen supply to the ischemic or injured tissue. Recent studies additionally suggest that the endothelium acts as a paracrine source for signals that determine tissue regeneration versus fibrosis after injury. Excessive vascularization, however, might also be unwanted, as in the case of cancer, neovascular eye diseases including diabetic retinopathy, atheroma growth, or the expansion of vasa vasorum, which leads to adverse vessel wall remodeling. Neovascularization is a tightly regulated and essential process that results in the formation of new blood vessels. Specific types of neovascularization include angiogenesis, the formation of new capillaries from existing capillaries, and arteriogenesis, the formation of new arteries from preexisting collaterals or de novo. Although endothelial cells (ECs) certainly are essential for both processes, the formation of functionally active vessels requires a complex molecular cross-talk of ECs with perivascular cells such as pericytes, smooth muscle cells, and macrophages. Simple in vitro models are best suited to examine specific aspects of particular processes involved in angiogenesis such as the biochemical interactions that regulate EC proliferation, motility, and apoptosis or lumen formation. However, in vivo models are required to understand the complex cellular interactions that enable the generation of functionally active and hierarchical blood vessel networks capable of providing an appropriate blood supply and paracrine stimuli to organs. In addition, the development of therapeutic strategies to either promote or inhibit vessel growth depends on reproducible measures and end points in experimental …

Patients with peripheral artery disease have a marked reduction in exercise performance and daily ambulatory activity irrespective of their limb symptoms of classic or atypical claudication. This review will evaluate the multiple pathophysiologic mechanisms underlying the exercise impairment in peripheral artery disease based on an evaluation of the current literature and research performed by the authors. Peripheral artery disease results in atherosclerotic obstructions in the major conduit arteries supplying the lower extremities. This arterial disease process impairs the supply of oxygen and metabolic substrates needed to match the metabolic demand generated by active skeletal muscle during walking exercise. However, the hemodynamic impairment associated with the occlusive disease process does not fully account for the reduced exercise impairment, indicating that additional pathophysiologic mechanisms contribute to the limb manifestations. These mechanisms include a cascade of pathophysiological responses during exercise-induced ischemia and reperfusion at rest that are associated with endothelial dysfunction, oxidant stress, inflammation, and muscle metabolic abnormalities that provide opportunities for targeted therapeutic interventions to address the complex pathophysiology of the exercise impairment in peripheral artery disease.

Fibrotic diseases are a significant global burden for which there are limited treatment options. The effector cells of fibrosis are activated fibroblasts called myofibroblasts, a highly contractile cell type characterized by the appearance of α-smooth muscle actin stress fibers. The underlying mechanism behind myofibroblast differentiation and persistence has been under much investigation and is known to involve a complex signaling network involving transforming growth factor-β, endothelin-1, angiotensin II, CCN2 (connective tissue growth factor), and platelet-derived growth factor. This review addresses the contribution of these signaling molecules to cardiac fibrosis.

Although numerous preclinical investigations have consistently demonstrated salubrious effects of c-kit pos cardiac cells administered after myocardial infarction, the mechanism of action remains highly controversial. We and others have found little or no evidence that these cells differentiate into mature functional cardiomyocytes, suggesting paracrine effects. In this review, we propose a new paradigm predicated on a comprehensive analysis of the literature, including studies of cardiac development; we have (facetiously) dubbed this conceptual construct “string theory” of c-kit pos cardiac cells because it reconciles multifarious and sometimes apparently discrepant results. There is strong evidence that, during development, the c-kit receptor is expressed in different pools of cardiac progenitors (some capable of robust cardiomyogenesis and others with little or no contribution to myocytes). Accordingly, c-kit positivity, in itself, does not define the embryonic origins, lineage capabilities, or differentiation capacities of specific cardiac progenitors. C-kit pos cells derived from the first heart field exhibit cardiomyogenic potential during development, but these cells are likely depleted shortly before or after birth. The residual c-kit pos cells found in the adult heart are probably of proepicardial origin, possess a mesenchymal phenotype (resembling bone marrow mesenchymal stem/stromal cells), and are capable of contributing significantly only to nonmyocytic lineages (fibroblasts, smooth muscle cells, and endothelial cells). If these 2 populations (first heart field and proepicardium) express different levels of c-kit, the cardiomyogenic potential of first heart field progenitors might be reconciled with recent results of c-kit pos cell lineage tracing studies. The concept that c-kit expression in the adult heart identifies epicardium-derived, noncardiomyogenic precursors with a mesenchymal phenotype helps to explain the beneficial effects of c-kit pos cell administration to ischemically damaged hearts despite the observed paucity of cardiomyogenic differentiation of these cells.

Until recently, significant advances in our understanding of the mechanisms of blood pressure regulation arose from studies of monogenic forms of hypertension and hypotension, which identified rare variants that primarily alter renal salt handling. Genome-wide association and exome sequencing studies over the past 6 years have resulted in an unparalleled burst of discovery in the genetics of blood pressure regulation and hypertension. More importantly, genome-wide association studies, while expanding the list of common genetic variants associated with blood pressure and hypertension, are also uncovering novel pathways of blood pressure regulation that augur a new era of novel drug development, repurposing, and stratification in the management of hypertension. In this review, we describe the current state of the art of the genetic and molecular basis of blood pressure and hypertension.

For >50 years, it has been recognized that immunity contributes to hypertension. Recent data have defined an important role of T cells and various T cell–derived cytokines in several models of experimental hypertension. These studies have shown that stimuli like angiotensin II, deoxycorticosterone acetate-salt, and excessive catecholamines lead to formation of effector like T cells that infiltrate the kidney and perivascular regions of both large arteries and arterioles. There is also accumulation of monocyte/macrophages in these regions. Cytokines released from these cells, including interleukin-17, interferon-γ, tumor necrosis factorα, and interleukin-6 promote both renal and vascular dysfunction and damage, leading to enhanced sodium retention and increased systemic vascular resistance. The renal effects of these cytokines remain to be fully defined, but include enhanced formation of angiotensinogen, increased sodium reabsorption, and increased renal fibrosis. Recent experiments have defined a link between oxidative stress and immune activation in hypertension. These have shown that hypertension is associated with formation of reactive oxygen species in dendritic cells that lead to formation of gamma ketoaldehydes, or isoketals. These rapidly adduct to protein lysines and are presented by dendritic cells as neoantigens that activate T cells and promote hypertension. Thus, cells of both the innate and adaptive immune system contribute to end-organ damage and dysfunction in hypertension. Therapeutic interventions to reduce activation of these cells may prove beneficial in reducing end-organ damage and preventing consequences of hypertension, including myocardial infarction, heart failure, renal failure, and stroke.

Rationale: Purinergic signaling plays an important role in inflammation and vascular integrity, but little is known about purinergic mechanisms during the pathogenesis of atherosclerosis in humans. Objective: The objective of this study is to study markers of purinergic signaling in a cohort of patients with peripheral artery disease. Methods and Results: Plasma ATP and ADP levels and serum nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39) and ecto-5′-nucleotidase/CD73 activities were measured in 226 patients with stable peripheral artery disease admitted for nonurgent invasive imaging and treatment. The major findings were that ATP, ADP, and CD73 values were higher in atherosclerotic patients than in controls without clinically evident peripheral artery disease ( P P =0.01). In multivariable linear regression models, high CD73 activity was associated with chronic hypoxia ( P =0.001). Statin use was associated with lower ADP ( P =0.041) and tended to associate with higher CD73 ( P =0.054), while lower ATP was associated with the use of angiotensin receptor blockers ( P =0.015). Conclusions: Purinergic signaling plays an important role in peripheral artery disease progression. Elevated levels of circulating ATP and ADP are especially associated with atherosclerotic diseases of younger age and smoking. The antithrombotic and anti-inflammatory effects of statins may partly be explained by their ability to lower ADP. We suggest that the prothrombotic nature of smoking could be a cause of elevated ADP, and this may explain why cardiovascular patients who smoke benefit from platelet P2Y 12 receptor antagonists more than their nonsmoking peers.

Fiber organization is important for myocardial excitation and contraction. It can be a major factor in arrhythmogenesis and current distribution during defibrillation shocks. In this study, we report the discovery of a previously undetected thin epicardial layer in swine right ventricle (RV) with distinctly different fiber orientation, which significantly affects epicardial propagation. Experiments were conducted in isolated coronary-perfused right ventricular free wall preparations (n=8) stained with the voltage-sensitive dye di-4-ANEPPS. Optical signals were recorded from the epicardium with a CCD video camera at 800 fps. Preparations were sectioned parallel to the epicardial surface with a resolution of 50 μm or better. To link the histological data with the observed activation patterns, resulting fiber angles were introduced into a 3D computer model to simulate the electrical activation and voltage-dependent optical signals. In all preparations, we detected a thin epicardial layer with almost no depth-dependent fiber rotation. The thickness of this layer ( z 0 ) varied from 110 to 930 μm. At the boundary of this layer, we observed an abrupt change in fiber angle by 64±13° followed by a gradual fiber rotation in the underlying layers. In preparations with z 0

The KCNQ1-G589D gene mutation, associated with a long-QT syndrome, has been shown to disrupt yotiao-mediated targeting of protein kinase A and protein phosphatase-1 to the I Ks channel. To investigate how this defect may lead to ventricular arrhythmia during sympathetic stimulation, we use integrative computational models of β-adrenergic signaling, myocyte excitation-contraction coupling, and action potential propagation in a rabbit ventricular wedge. Paradoxically, we find that the KCNQ1-G589D mutation alone does not prolong the QT interval. But when coupled with β-adrenergic stimulation in a whole-cell model, the KCNQ1-G589D mutation induced QT prolongation and transient afterdepolarizations, known cellular mechanisms for arrhythmogenesis. These cellular mechanisms amplified tissue heterogeneities in a three-dimensional rabbit ventricular wedge model, elevating transmural dispersion of repolarization and creating other T-wave abnormalities on simulated electrocardiograms. Increasing heart rate protected both single myocyte and the coupled myocardium models from arrhythmic consequences. These findings suggest that the KCNQ1-G589D mutation disrupts a critical link between β-adrenergic signaling and myocyte electrophysiology, creating both triggers of cardiac arrhythmia and a myocardial substrate vulnerable to such electrical disturbances.

Several studies implicate elevated matrix metalloproteinase activity as a cause of cardiac fibrosis. However, it is unknown whether other proteases can also initiate cardiac fibrosis. Because absence of urokinase plasminogen activator (uPA) prevents development of cardiac fibrosis after experimental myocardial infarction in mice, we hypothesized that elevated activity of uPA or deficiency of the uPA inhibitor plasminogen activator inhibitor-1 (PAI-1) might cause cardiac fibrosis. We used mice with scavenger-receptor (SR)-directed, macrophage-targeted uPA overexpression (SR-uPA +/0 mice) and PAI-1 null mice to test these hypotheses. Our studies revealed that SR-uPA +/0 mice developed cardiac fibrosis beginning between 5 and 10 weeks of age. Fibrosis was preceded by cardiac macrophage accumulation, implicating uPA-secreting macrophages as important contributors to development of fibrosis. A key role for uPA-secreting macrophages in development of cardiac fibrosis was supported by experiments in which recipients of bone marrow transplants from SR-uPA +/0 donors but not nontransgenic donors developed cardiac macrophage accumulation and fibrosis. SR-uPA +/0 mice and recipients of SR-uPA +/0 bone marrow had neither macrophage accumulation nor fibrosis in other major organs despite the presence of higher levels of uPA in these organs than in hearts. PAI-1 null mice but not congenic, age-matched controls also developed macrophage accumulation and fibrosis in hearts but not in other organs. We conclude: (1) either elevated macrophage uPA expression or PAI-1 deficiency is sufficient to cause cardiac macrophage accumulation and fibrosis; (2) macrophages are important contributors to the development of cardiac fibrosis; and (3) the heart is particularly sensitive to the effects of excess uPA activity.

During the past decade, single gene disruption in mice and large-scale mutagenesis screens in zebrafish have elucidated many fundamental genetic pathways that govern early heart patterning and differentiation. Specifically, a number of genes have been revealed serendipitously to play important and selective roles in cardiac valve development. These initially surprising results have now converged on a finite number of signaling pathways that regulate endothelial proliferation and differentiation in developing and postnatal heart valves. This review highlights the roles of the most well-established ligands and signaling pathways, including VEGF, NFATc1, Notch, Wnt/β-catenin, BMP/TGF-β, ErbB, and NF1/Ras. Based on the interactions among and relative timing of these pathways, a signaling network model for heart valve development is proposed.

Growth of collateral blood vessels (arteriogenesis) is potentially able to preserve structure and function of limbs and organs after occlusion of a major artery. The success of the remodeling process depends on the following conditions: (1) existence of an arteriolar network that connects the preocclusive with the postocclusive microcirculation; (2) activation of the arteriolar endothelium by elevated fluid shear stress; (3) invasion (but not incorporation) of bone marrow–derived cells; and (4) proliferation of endothelial and smooth muscle cells. Most organs of most mammals including man can rely on the existence of interconnecting arterioles in most organs and tissues with heart being the exception in rodents and pigs. Arterial occlusion lowers the pressure in the distal vasculature thereby creating a pressure gradient favoring increased flow through preexisting collaterals. This increases fluid shear stress leading to endothelial activation with cellular edema, upregulation of adhesion molecules, mitogenic-, thrombogenic-, and fibrinolytic factors, leading to monocyte invasion with matrix digestion. Smooth muscle cells migrate and proliferate and the vessel enlarges under the influence of increasing circumferential wall stress. Growth factors involved belong to the FGF family and signaling proceeds via the Ras/Raf- and the Rho cascades. Increases in vascular radius and wall thickness restore fluid shear stress and circumferential wall stress to normal levels and growth stops. Although increases in collateral vessel size are very substantial their maximal conductance amounts to only 40% of normal. Forced increases in FSS can reach almost 100%.

Previous modeling studies have suggested that the rapid component of the delayed rectifier ( I Kr ) may contribute importantly to action potential dynamics during tachycardia. To test this idea experimentally, I Kr was measured as the E-4031–sensitive current in isolated canine endocardial myocytes at 37°C using the perforated patch-clamp technique. Command potentials were trains of action potential waveforms recorded at cycle lengths (CLs) of 1000, 500, 320, 170, and 120 ms. Action potential duration (APD) alternans occurred at CLs of 170 and 120 ms. During an action potential, I Kr increased gradually to a maximum at −55 to −60 mV. Peak I Kr increased initially as CL was shortened from 1000 to 500 ms (from 0.55±0.03 to 0.57±0.03 pA/pF), but decreased progressively as CL was shortened further (to 0.45±0.03 pA/pF at CL=120 ms). Baseline I Kr was negligible at CLs of 1000 to 320 ms, but increased to 0.12±0.01 pA/pF at a CL of 120 ms. During APD alternans, peak I Kr was larger for the short than for the long action potential (0.48±0.03 versus 0.46±0.03 pA/pF). A computer model of I Kr based on these data indicated that increasing I Kr suppressed alternans and decreasing I Kr increased alternans. In support of the latter result, inhibition of I Kr by E-4031 increased the maximal amplitude of alternans. These results indicate that I Kr contributes importantly to rate-related alterations of repolarization, including APD alternans. Modifying I Kr may be a promising approach to suppressing alternans and thereby preventing ventricular tachyarrhythmias.

Interruption of periodic wave propagation by the nucleation and subsequent disintegration of spiral waves is thought to mediate the transition from normal sinus rhythm to ventricular fibrillation. This sequence of events may be precipitated by a period doubling bifurcation, manifest as a beat-to-beat alternation, or alternans, of cardiac action potential duration and conduction velocity. How alternans causes the local conduction block required for initiation of spiral wave reentry remains unclear, however. In the present study, a mechanism for conduction block was derived from experimental studies in linear strands of cardiac tissue and from computer simulations in ionic and coupled maps models of homogeneous one-dimensional fibers. In both the experiments and the computer models, rapid periodic pacing induced marked spatiotemporal heterogeneity of cellular electrical properties, culminating in paroxysmal conduction block. These behaviors resulted from a nonuniform distribution of action potential duration alternans, secondary to alternans of conduction velocity. This link between period doubling bifurcations of cellular electrical properties and conduction block may provide a generic mechanism for the onset of tachycardia and fibrillation.