EHD3-dependent endosome pathway regulates cardiac membrane excitability and physiology.

RATIONALE: Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology. OBJECTIVE: To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology. METHODS AND RESULTS: We identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger-mediated membrane current and Cav1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes. CONCLUSIONS: These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.

Upregulation of adenosine A1 receptors facilitates sinoatrial node dysfunction in chronic canine heart failure by exacerbating nodal conduction abnormalities revealed by novel dual-sided intramural optical mapping.

BACKGROUND: Although sinoatrial node (SAN) dysfunction is a hallmark of human heart failure (HF), the underlying mechanisms remain poorly understood. We aimed to examine the role of adenosine in SAN dysfunction and tachy-brady arrhythmias in chronic HF. METHODS AND RESULTS: We applied multiple approaches to characterize SAN structure, SAN function, and adenosine A1 receptor expression in control (n=17) and 4-month tachypacing-induced chronic HF (n=18) dogs. Novel intramural optical mapping of coronary-perfused right atrial preparations revealed that adenosine (10 µmol/L) markedly prolonged postpacing SAN conduction time in HF by 206 ± 99 milliseconds (versus 66 ± 21 milliseconds in controls; P=0.02). Adenosine induced SAN intranodal conduction block or microreentry in 6 of 8 dogs with HF versus 0 of 7 controls (P=0.007). Adenosine-induced SAN conduction abnormalities and automaticity depression caused postpacing atrial pauses in HF versus control dogs (17.1 ± 28.9 versus 1.5 ± 1.3 seconds; P<0.001). Furthermore, 10 µmol/L adenosine shortened atrial repolarization and led to pacing-induced atrial fibrillation in 6 of 7 HF versus 0 of 7 control dogs (P=0.002). Adenosine-induced SAN dysfunction and atrial fibrillation were abolished or prevented by adenosine A1 receptor antagonists (50 µmol/L theophylline/1 µmol/L 8-cyclopentyl-1,3-dipropylxanthine). Adenosine A1 receptor protein expression was significantly upregulated during HF in the SAN (by 47 ± 19%) and surrounding atrial myocardium (by 90 ± 40%). Interstitial fibrosis was significantly increased within the SAN in HF versus control dogs (38 ± 4% versus 23 ± 4%; P<0.001). CONCLUSIONS: In chronic HF, adenosine A1 receptor upregulation in SAN pacemaker and atrial cardiomyocytes may increase cardiac sensitivity to adenosine. This effect may exacerbate conduction abnormalities in the structurally impaired SAN, leading to SAN dysfunction, and potentiate atrial repolarization shortening, thereby facilitating atrial fibrillation. Atrial fibrillation may further depress SAN function and lead to tachy-brady arrhythmias in HF.

Shortened Ca2+ signaling refractoriness underlies cellular arrhythmogenesis in a postinfarction model of sudden cardiac death.

RATIONALE: Diastolic spontaneous Ca(2+) waves (DCWs) are recognized as important contributors to triggered arrhythmias. DCWs are thought to arise when [Ca(2+)] in sarcoplasmic reticulum ([Ca(2+)](SR)) reaches a certain threshold level, which might be reduced in cardiac disease as a consequence of sensitization of ryanodine receptors (RyR2s) to luminal Ca(2+). OBJECTIVE: We investigated the mechanisms of DCW generation in myocytes from normal and diseased hearts, using a canine model of post-myocardial infarction ventricular fibrillation (VF). METHODS AND RESULTS: The frequency of DCWs, recorded during periodic pacing in the presence of a ß-adrenergic receptor agonist isoproterenol, was significantly higher in VF myocytes than in normal controls. Rather than occurring immediately on reaching a final [Ca(2+)](SR), DCWs arose with a distinct time delay after attaining steady [Ca(2+)](SR) in both experimental groups. Although the rate of [Ca(2+)](SR) recovery after the SR Ca(2+) release was similar between the groups, in VF myocytes the latency to DCWs was shorter, and the [Ca(2+)](SR) at DCW initiation was lower. The restitution of depolarization-induced Ca(2+) transients, assessed by a 2-pulse protocol, was significantly faster in VF myocytes than in controls. The VF-related alterations in myocyte Ca(2+) cycling were mimicked by the RyR2 agonist, caffeine. The reducing agent, mercaptopropionylglycine, or the CaMKII inhibitor, KN93, decreased DCW frequency and normalized restitution of Ca(2+) release in VF myocytes. CONCLUSIONS: The attainment of a certain threshold [Ca(2+)](SR) is not sufficient for the generation of DCWs. Postrelease Ca(2+) signaling refractoriness critically influences the occurrence of DCWs. Shortened Ca(2+) signaling refractoriness due to RyR2 phosphorylation and oxidation is responsible for the increased rate of DCWs observed in VF myocytes and could provide a substrate for synchronization of arrhythmogenic events at the tissue level in hearts prone to VF.

Neuronal nitric oxide synthase is indispensable for the cardiac adaptive effects of exercise.

Exercise results in beneficial adaptations of the heart that can be directly observed at the ventricular myocyte level. However, the molecular mechanism(s) responsible for these adaptations are not well understood. Interestingly, signaling via neuronal nitric oxide synthase (NOS1) within myocytes results in similar effects as exercise. Thus, the objective was to define the role NOS1 plays in the exercise-induced beneficial contractile effects in myocytes. After an 8-week aerobic interval training program, exercise-trained (Ex) mice had higher VO(2max) and cardiac hypertrophy compared to sedentary (Sed) mice. Ventricular myocytes from Ex mice had increased NOS1 expression and nitric oxide production compared to myocytes from Sed mice. Remarkably, acute NOS1 inhibition normalized the enhanced contraction (shortening and Ca(2+) transients) in Ex myocytes to Sed levels. The NOS1 effect on contraction was mediated via greater Ca(2+) cycling that resulted from increased phospholamban phosphorylation. Intriguingly, a similar aerobic interval training program on NOS1 knockout mice failed to produce any beneficial cardiac adaptations (VO(2max), hypertrophy, and contraction). These data demonstrate that the beneficial cardiac adaptations observed after exercise training were mediated via enhanced NOS1 signaling. Therefore, it is likely that beneficial effects of exercise may be mimicked by the interventions that increase NOS1 signaling. This pathway may provide a potential novel therapeutic target in cardiac patients who are unable or unwilling to exercise.

At-risk but viable myocardium in a large animal model of non ST-segment elevation acute coronary syndrome: cardiovascular magnetic resonance with ex vivo validation.

BACKGROUND: Patients with non-ST-segment elevation acute coronary syndrome (NSTE-ACS) have varying degrees of salvageable myocardium at risk of irreversible injury. We hypothesized that a novel model of NSTE-ACS produces acute myocardial injury, measured by increased T2 cardiovascular magnetic resonance (CMR), without significant necrosis by late gadolinium enhancement (LGE). METHODS: In a canine model, partial coronary stenosis was created and electrodes placed on the epicardium. Myocardial T2, an indicator of at-risk myocardium, was measured pre- and post-tachycardic pacing. RESULTS: Serum troponin-I (TnI) was not detectable in unoperated sham animals but averaged 1.97 ± 0.72 ng/mL in model animals. Coronary stenosis and pacing produced significantly higher T2 in the affected vs. the remote myocardium (53.2 ± 4.9 vs. 43.6 ± 2.8 ms, p < 0.01) with no evident injury by LGE. Microscopy revealed no significant irreversible cellular injury. Relative respiration rate (RRR) of affected vs. remote myocardial tissue was significantly lower in model vs. sham animals (0.72 ± 0.07 vs. 1.04 ± 0.07, p < 0.001). Lower RRR corresponded to higher final TnI levels (R(2) = 0.83, p = 0.004) and changes in CaMKIID and mitochondrial gene expression. CONCLUSIONS: A large animal NSTE-ACS model with mild TnI elevation and without ST elevation, similar to the human syndrome, demonstrates signs of acute myocardial injury by T2-CMR without significant irreversible damage. Reduced tissue respiration and associated adaptations of critical metabolic pathways correspond to increased myocardial injury by serum biomarkers in this model. T2-CMR as a biomarker of at-risk but salvageable myocardium warrants further consideration in preclinical and clinical studies of NSTE-ACS.

Effect of dietary omega-3 fatty acids on the heart rate and the heart rate variability responses to myocardial ischemia or submaximal exercise.

The consumption of omega-3 polyunsaturated fatty acids (n-3 PUFAs) has been reported to decrease resting heart rate (HR) and increase heart rate variability (HRV). However, the effects of n-3 PUFAs on these variables in response to a physiological stress (e.g., exercise or acute myocardial ischemia), particularly in postmyocardial infarction (MI) patients, are unknown. Therefore, HR and HRV (high frequency and total R-R interval variability) were evaluated at rest, during submaximal exercise, and during a 2-min coronary artery occlusion at rest and before and 3 mo after n-3 PUFA treatment in dogs with healed MI (n = 59). The dogs were randomly assigned to either placebo (1 g/day corn oil, n = 19) or n-3 PUFA supplement (docosahexaenoic acid + eicosapentaenoic acid ethyl esters; 1 g/day, n = 6; 2 g/day, n = 12; or 4 g/day, n = 22) groups. The treatment elicited significant (P < 0.01) dose-dependent increases in right atrial n-3 PUFA levels but dose-independent reductions in resting HR and increases in resting HRV. In contrast, n-3 PUFAs did not attenuate the large changes in HR or HRV induced by either the coronary occlusion or submaximal exercise. These data demonstrate that dietary n-3 PUFA decreased resting (i.e., preexercise or preocclusion) HR and increased resting HRV but did not alter the cardiac response to physiologic challenges.

Contraction-relaxation coupling is unaltered by exercise training and infarction in isolated canine myocardium.

The two main phases of the mammalian cardiac cycle are contraction and relaxation; however, whether there is a connection between them in humans is not well understood. Routine exercise has been shown to improve cardiac function, morphology, and molecular signatures. Likewise, the acute and chronic changes that occur in the heart in response to injury, disease, and stress are well characterized, albeit not fully understood. In this study, we investigated how exercise and myocardial injury affect contraction-relaxation coupling. We retrospectively analyzed the correlation between the maximal speed of contraction and the maximal speed of relaxation of canine myocardium after receiving surgically induced myocardial infarction, followed by either sedentary recovery or exercise training for 10-12 wk. We used isolated right ventricular trabeculae, which were electrically paced at different lengths, frequencies, and with increasing ß-adrenoceptor stimulation. In all conditions, contraction and relaxation were linearly correlated, irrespective of injury or training history. Based on these results and the available literature, we posit that contraction-relaxation coupling is a fundamental myocardial property that resides in the structural arrangement of proteins at the level of the sarcomere and that this may be regulated by the actions of cardiac myosin binding protein C (cMyBP-C) on actin and myosin.

Effects of dietary omega-3 fatty acids on ventricular function in dogs with healed myocardial infarctions: in vivo and in vitro studies.

Since omega-3 polyunsaturated fatty acids (n-3 PUFAs) can alter ventricular myocyte calcium handling, these fatty acids could adversely affect cardiac contractile function, particularly following myocardial infarction. Therefore, 4 wk after myocardial infarction, dogs were randomly assigned to either placebo (corn oil, 1 g/day, n = 16) or n-3 PUFAs supplement [docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) ethyl esters; 1, 2, or 4 g/day; n = 7, 8, and 12, respectively] groups. In vivo, ventricular function was evaluated by echocardiography before and after 3 mo of treatment. At the end of the 3-mo period, hearts were removed and in vitro function was evaluated using right ventricular trabeculae and isolated left ventricular myocytes. The treatment elicited significant (P < 0.0001) dose-dependent increases (16.4-fold increase with 4 g/day) in left ventricular tissue and red blood cell n-3 PUFA levels (EPA + DHA, placebo, 0.42 +/- 0.04; 1 g/day, 3.02 +/- 0.23; 2 g/day, 3.63 +/- 0.17; and 4 g/day, 6.97 +/- 0.33%). Regardless of the dose, n-3 PUFA treatment did not alter ventricular function in the intact animal (e.g., 4 g/day, fractional shortening: pre, 42.9 +/- 1.6 vs. post, 40.1 +/- 1.7%; placebo: pre, 39.2 +/- 1.3 vs. post, 38.4 +/- 1.6%). The developed force per cross-sectional area, changes in length- and frequency-dependent behavior in contractile force, and the inotropic response to beta-adrenoceptor activation were also similar for trabeculae obtained from placebo- or n-3 PUFA-treated dogs. Finally, calcium currents and calcium transients were the same in myocytes from n-3 PUFA- and placebo-treated dogs. Thus dietary n-3 PUFAs did not adversely alter either in vitro or in vivo ventricular contractile function in dogs with healed infarctions.

Homeostasis: The Underappreciated and Far Too Often Ignored Central Organizing Principle of Physiology.

The grand challenge to physiology, as was first described in an essay published in the inaugural issue of Frontiers in Physiology in 2010, remains to integrate function from molecules to intact organisms. In order to make sense of the vast volume of information derived from, and increasingly dependent upon, reductionist approaches, a greater emphasis must be placed on the traditional integrated and more holistic approaches developed by the scientists who gave birth to physiology as an intellectual discipline. Our understanding of physiological regulation has evolved over time from the Greek idea of body humors, through Claude Bernard's "milieu intérieur," to Walter Cannon's formulation of the concept of "homeostasis" and the application of control theory (feedback and feedforward regulation) to explain how a constant internal environment is achieved. Homeostasis has become the central unifying concept of physiology and is defined as a self-regulating process by which an organism can maintain internal stability while adjusting to changing external conditions. Homeostasis is not static and unvarying; it is a dynamic process that can change internal conditions as required to survive external challenges. It is also important to note that homeostatic regulation is not merely the product of a single negative feedback cycle but reflects the complex interaction of multiple feedback systems that can be modified by higher control centers. This hierarchical control and feedback redundancy results in a finer level of control and a greater flexibility that enables the organism to adapt to changing environmental conditions. The health and vitality of the organism can be said to be the end result of homeostatic regulation. An understanding of normal physiology is not possible without an appreciation of this concept. Conversely, it follows that disruption of homeostatic mechanisms is what leads to disease, and effective therapy must be directed toward re-establishing these homeostatic conditions. Therefore, it is the purpose of this essay to describe the evolution of our understanding of homeostasis and the role of physiological regulation and dysregulation in health and disease.

Signatures of the autonomic nervous system and the heart's pacemaker cells in canine electrocardiograms and their applications to humans.

Heart rate and heart rate variability (HRV) are mainly determined by the autonomic nervous system (ANS), which interacts with receptors on the sinoatrial node (SAN; the heart's primary pacemaker), and by the "coupled-clock" system within the SAN cells. HRV changes are associated with cardiac diseases. However, the relative contributions of the ANS and SAN to HRV are not clear, impeding effective treatment. To discern the SAN's contribution, we performed HRV analysis on canine electrocardiograms containing basal and ANS-blockade segments. We also analyzed human electrocardiograms of atrial fibrillation and heart failure patients, as well as healthy aged subjects. Finally, we used a mathematical model to simulate HRV under decreased "coupled-clock" regulation. We found that (a) in canines, the SAN and ANS contribute mainly to long- and short-term HRV, respectively; (b) there is evidence suggesting a similar relative SAN contribution in humans; (c) SAN features can be calculated from beat-intervals obtained in-vivo, without intervention; (d) ANS contribution can be modeled by sines embedded in white noise; (e) HRV changes associated with cardiac diseases and aging can be interpreted as deterioration of both SAN and ANS; and (f) SAN clock-coupling can be estimated from changes in HRV. This may enable future non-invasive diagnostic applications.

Cardiac autonomic neural remodeling and susceptibility to sudden cardiac death: effect of endurance exercise training.

Sudden cardiac death resulting from ventricular tachyarrhythmias remains the leading cause of death in industrially developed countries, accounting for between 300,000 and 500,000 deaths each year in the United States. Yet, despite the enormity of this problem, both the identification of factors contributing to ventricular fibrillation as well as the development of safe and effective antiarrhythmic agents remain elusive. Subnormal cardiac parasympathetic regulation coupled with an elevated cardiac sympathetic activation may allow for the formation of malignant ventricular arrhythmias. In particular, myocardial infarction can reduce cardiac parasympathetic regulation and alter beta-adrenoceptor subtype expression enhancing beta(2)-adrenoceptor sensitivity that can lead to intracellular calcium dysregulation and arrhythmias. As such, myocardial infarction can induce a remodeling of cardiac autonomic regulation that may be required to maintain cardiac pump function. If alterations in cardiac autonomic regulation play an important role in the genesis of life-threatening arrhythmias, then one would predict that interventions designed to either augment parasympathetic activity and/or reduce cardiac adrenergic activity would also protect against ventricular fibrillation. Recently, studies using a canine model of sudden death demonstrate that endurance exercise training (treadmill running) enhanced cardiac parasympathetic regulation (increased heart rate variability), restored a more normal beta-adrenoceptor balance (i.e., reduced beta(2)-adrenoceptor sensitivity and expression), and protected against ventricular fibrillation induced by acute myocardial ischemia. Thus exercise training may reverse the autonomic neural remodeling induced by myocardial infarction and thereby enhance the electrical stability of the heart in individuals shown to be at an increased risk for sudden cardiac death.

The cardiac sarcolemmal ATP-sensitive potassium channel as a novel target for anti-arrhythmic therapy.

The activation of cardiac cell membrane ATP-sensitive potassium channels during myocardial ischemia promotes potassium efflux, reductions in action potential duration, and heterogeneities in repolarization, thereby creating a substrate for re-entrant arrhythmias. Drugs that block this channel should be particularly effective anti-arrhythmic agents. Indeed, non-selective ATP-sensitive potassium channel antagonists, (e.g., glibenclamide) can prevent arrhythmias associated with myocardial ischemia. However, these non-selective antagonists have important non-cardiac actions that promote insulin release and hypoglycemia (pancreatic beta-cells), reduce coronary blood flow (vascular smooth muscle cells), prevent ischemia preconditioning (cardiac mitochondrial channels) and depress cardiac contractile function. The ATP-sensitive potassium channel consists of a pore forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and a regulatory subunit (sulfonylurea receptors, SUR1, SUR2A &SUR2B). The Kir6.2/SUR2A combination appears to be preferentially expressed on cardiac cell membranes. As such, it should be possible to develop agents selective for cardiac sarcolemmal ATP-sensitive potassium channels. The novel compounds HMR 1883 (or its sodium salt HMR 1098) or HMR 1402 have been shown to block selectively the cardiac sarcolemmal ATP-sensitive potassium channels. These drugs attenuated ischemically-induced changes in cardiac electrical properties and prevented malignant arrhythmias without the untoward effects of other drugs. Since the ATP-sensitive potassium channel only becomes active as ATP levels fall, these drugs have the added advantage that they would have effects only on ischemic tissue with little or no effect noted on normal tissue. Thus, selective antagonists of the cardiac cell surface ATP-sensitive potassium channel may represent a new class of ischemia selective anti-arrhythmic medications.

Electrotonic remodeling following myocardial infarction in dogs susceptible and resistant to sudden cardiac death.

Passive electrical remodeling following myocardial infarction (MI) is well established. These changes can alter electrotonic loading and trigger the remodeling of repolarization currents, a potential mechanism for ventricular fibrillation (VF). However, little is known about the role of passive electrical markers as tools to identify VF susceptibility post-MI. This study investigated electrotonic remodeling in the post-MI ventricle, as measured by myocardial electrical impedance (MEI), in animals prone to and resistant to VF. MI was induced in dogs by a two-stage left anterior descending (LAD) coronary artery ligation. Before infarction, MEI electrodes were placed in remote (left circumflex, LCX) and infarcted (LAD) myocardium. MEI was measured in awake animals 1, 2, 7, and 21 days post-MI. Subsequently, VF susceptibility was tested by a 2-min LCX occlusion during exercise; 12 animals developed VF (susceptible, S) and 12 did not (resistant, R). The healing infarct had lower MEI than the normal myocardium. This difference was stable by day 2 post-MI (287 +/- 32 Omega vs. 425 +/- 62 Omega, P < 0.05). Significant differences were observed between resistant and susceptible animals 7 days post-MI; susceptible dogs had a wider electrotonic gradient between remote and infarcted myocardium (R: 89 +/- 60 Omega vs. S: 180 +/- 37 Omega). This difference increased over time in susceptible animals (252 +/- 53 Omega at 21 days) due to post-MI impedance changes on the remote myocardium. These data suggest that early electrotonic changes post-MI could be used to assess later arrhythmia susceptibility. In addition, passive-electrical changes could be a mechanism driving active-electrical remodeling post-MI, thereby facilitating the induction of arrhythmias.

Inflammation-Nature's Way to Efficiently Respond to All Types of Challenges: Implications for Understanding and Managing "the Epidemic" of Chronic Diseases.

Siloed or singular system approach to disease management is common practice, developing out of traditional medical school education. Textbooks of medicine describe a huge number of discrete diseases, usually in a systematic fashion following headings like etiology, pathology, investigations, differential diagnoses, and management. This approach suggests that the body has a multitude of ways to respond to harmful incidences. However, physiology and systems biology provide evidence that there is a simple mechanism behind this phenotypical variability. Regardless if an injury or change was caused by trauma, infection, non-communicable disease, autoimmune disorders, or stress, the typical physiological response is: an increase in blood supply to the area, an increase in white cells into the affected tissue, an increase in phagocytic activity to remove the offending agent, followed by a down-regulation of these mechanisms resulting in healing. The cascade of inflammation is the body's unique mechanism to maintain its integrity in response to macroscopic as well as microscopic injuries. We hypothesize that chronic disease development and progression are linked to uncontrolled or dysfunctional inflammation to injuries regardless of their nature, physical, environmental, or psychological. Thus, we aim to reframe the prevailing approach of management of individual diseases into a more integrated systemic approach of treating the "person as a whole," enhancing the patient experience, ability to a make necessary changes, and maximize overall health and well-being. The first part of the paper reviews the local immune cascades of pro- and anti-inflammatory regulation and the interconnected feedback loops with neural and psychological pathways. The second part emphasizes one of nature's principles at work-system design and efficiency. Continually overwhelming this finely tuned system will result in systemic inflammation allowing chronic diseases to emerge; the pathways of several common conditions are described in detail. The final part of the paper considers the implications of these understandings for clinical care and explore how this lens could shape the physician-patient encounter and health system redesign. We conclude that healthcare professionals must advocate for an anti-inflammatory lifestyle at the patient level as well as at the local and national levels to enhance population health and well-being.

A Universal Scaling Relation for Defining Power Spectral Bands in Mammalian Heart Rate Variability Analysis.

Background: Power spectral density (PSD) analysis of the heartbeat intervals in the three main frequency bands [very low frequency (VLF), low frequency (LF), and high frequency (HF)] provides a quantitative non-invasive tool for assessing the function of the cardiovascular control system. In humans, these frequency bands were standardized following years of empirical evidence. However, no quantitative approach has justified the frequency cutoffs of these bands and how they might be adapted to other mammals. Defining mammal-specific frequency bands is necessary if the PSD analysis of the HR is to be used as a proxy for measuring the autonomic nervous system activity in animal models. Methods: We first describe the distribution of prominent frequency peaks found in the normalized PSD of mammalian data using a Gaussian mixture model while assuming three components corresponding to the traditional VLF, LF and HF bands. We trained the algorithm on a database of human electrocardiogram recordings (n = 18) and validated it on databases of dogs (n = 17) and mice (n = 8). Finally, we tested it to predict the bands for rabbits (n = 4) for the first time. Results: Double-logarithmic analysis demonstrates a scaling law between the GMM-identified cutoff frequencies and the typical heart rate (HRm): fVLF-LF = 0.0037⋅ HRm0.58 , fLF-HF = 0.0017⋅ HRm1.01 and fHFup = 0.0128⋅ HRm0.86 . We found that the band cutoff frequencies and Gaussian mean scale with a power law of 1/4 or 1/8 of the typical body mass (BMm), thus revealing allometric power laws. Conclusion: Our automated data-driven approach allowed us to define the frequency bands in PSD analysis of beat-to-beat time series from different mammals. The scaling law between the band frequency cutoffs and the HRm can be used to approximate the PSD bands in other mammals.

Accentuated vagal antagonism paradoxically increases ryanodine receptor calcium leak in long-term exercised Calsequestrin2 knockout mice.

BACKGROUND: Long-term aerobic exercise alters autonomic balance, which may not be favorable in heart rate (HR)-dependent arrhythmic diseases including catecholaminergic polymorphic ventricular tachycardia (CPVT) because of preexisting bradycardia and increased sensitivity to parasympathetic stimulation. OBJECTIVE: The purpose of this study was to determine whether long-term exercise-induced autonomic adaptations modify CPVT susceptibility. METHODS: We determined exercise-induced parasympathetic effects on HR, arrhythmia incidence, and intracellular sarcoplasmic reticulum (SR) Ca2+ leak in atrial (ACM) and ventricular (VCM) cardiomyocytes, in exercised (EX) calsequestrin knockout (CASQ2-/-) mice, a model of CPVT. RESULTS: Although 8-week treadmill running improved exercise capacity in EX CPVT mice, the incidence and duration of ventricular tachycardia also increased. HR variability analyses revealed an increased high-frequency component of the power spectrum and root mean square of successive differences in R-R intervals indicating accentuated vagal antagonism during ß-adrenergic stimulation resulting in negligible HR acceleration. In EX CASQ2-/- VCM, peak amplitude of Ca2+ transient (CaT) increased, whereas SR Ca2+ content decreased. Aberrant Ca2+ sparks occurred at baseline, which was exacerbated with isoproterenol. Notably, although 10 µM of the cholinergic agonist carbachol prevented isoproterenol-induced Ca2+ waves in ACM, CaT amplitude, SR Ca2+ load, and isoproterenol-induced Ca2+ waves paradoxically increased in VCM. In parallel, ventricular ryanodine receptor (RyR2) protein expression increased, whereas protein kinase A- and calmodulin-dependent protein kinase II-mediated phosphorylation of RyR2 was not significantly altered, which could imply an increased number of "leaky" channels. CONCLUSION: Our novel results suggest that long-term exercise in CASQ2-/- mice increases susceptibility to ventricular arrhythmias by accentuating vagal antagonism during ß-adrenergic challenge, which prevents HR acceleration and exacerbates abnormal RyR2 Ca2+ leak in EX CASQ2-/- VCM.

A comprehensive review and analysis of 25 years of data from an in vivo canine model of sudden cardiac death: implications for future anti-arrhythmic drug development.

Sudden cardiac death resulting from ventricular tachyarrhythmias remains the leading cause of death in industrially developed countries, accounting for between 300,000 and 500,000 deaths each year in the United States. Yet, despite the enormity of this problem, the development of safe and effective anti-arrhythmic agents remains elusive. The identification of effective anti-arrhythmic agents is critically dependent upon the use of appropriate animal models of human disease. During the last 25 years, a canine model of sudden cardiac death has proven to be useful in both the identification of factors contributing to ventricular fibrillation (VF) and the evaluation of potential anti-arrhythmic therapies. The present review provides a detailed retrospective analysis of the data obtained with this model. Briefly, VF was reliably and reproducibly induced by the combination of acute myocardial ischemia at site distant from a previous myocardial infarction during submaximal exercise (to activate the autonomic nervous system). This exercise plus ischemia test identified 2 stable populations of dogs: those that development malignant arrhythmias (susceptible, n=303) and those that rarely developed even single premature ventricular activation (resistant, n=209). The susceptible animals exhibited an elevated sympathetic activation (due to an enhanced beta2-adrenoceptor responsiveness) and a subnormal parasympathetic regulation. Several interventions have proven to be particularly effective in preventing VF in the susceptible dogs; including calcium channel antagonists, left stellate ganglion disruption, ATP-sensitive potassium channel antagonists, beta-adrenoceptor antagonists, and non-pharmacological interventions (endurance exercise training and dietary omega-3 fatty acids).

Effect of endurance exercise training on heart rate onset and heart rate recovery responses to submaximal exercise in animals susceptible to ventricular fibrillation.

Both a large heart rate (HR) increase at exercise onset and a slow heart rate (HR) recovery following the termination of exercise have been linked to an increased risk for ventricular fibrillation (VF) in patients with coronary artery disease. Endurance exercise training can alter cardiac autonomic regulation. Therefore, it is possible that this intervention could restore a more normal HR regulation in high-risk individuals. To test this hypothesis, HR and HR variability (HRV, 0.24- to 1.04-Hz frequency component; an index of cardiac vagal activity) responses to submaximal exercise were measured 30, 60, and 120 s after exercise onset and 30, 60, and 120 s following the termination of exercise in dogs with healed myocardial infarctions known to be susceptible (n = 19) to VF (induced by a 2-min coronary occlusion during the last minute of a submaximal exercise test). These studies were then repeated after either a 10-wk exercise program (treadmill running, n = 10) or an equivalent sedentary period (n = 9). After 10 wk, the response to exercise was not altered in the sedentary animals. In contrast, endurance exercise increased indexes of cardiac vagal activity such that HR at exercise onset was reduced (30 s after exercise onset: HR pretraining 179 +/- 8.4 vs. posttraining 151.4 +/- 6.6 beats/min; HRV pretraining 4.0 +/- 0.4 vs. posttraining 5.8 +/- 0.4 ln ms(2)), whereas HR recovery 30 s after the termination of exercise increased (HR pretraining 186 +/- 7.8 vs. posttraining 159.4 +/- 7.7 beats/min; HRV pretraining 2.4 +/- 0.3 vs. posttraining 4.0 +/- 0.6 ln ms(2)). Thus endurance exercise training restored a more normal HR regulation in dogs susceptible to VF.

Effects of endurance exercise training on heart rate variability and susceptibility to sudden cardiac death: protection is not due to enhanced cardiac vagal regulation.

Low heart rate variability (HRV) is associated with an increased susceptibility to ventricular fibrillation (VF). Exercise training can increase HRV (an index of cardiac vagal regulation) and could, thereby, decrease the risk for VF. To test this hypothesis, a 2-min coronary occlusion was made during the last min of a 18-min submaximal exercise test in dogs with healed myocardial infarctions; 20 had VF (susceptible), and 13 did not (resistant). The dogs then received either a 10-wk exercise program (susceptible, n=9; resistant, n=8) or an equivalent sedentary period (susceptible, n=11; resistant, n=5). HRV was evaluated at rest, during exercise, and during a 2-min occlusion at rest and before and after the 10-wk period. Pretraining, the occlusion provoked significantly (P<0.01) greater increases in HR (susceptible, 54.9+/-8.3 vs. resistant, 25.0+/-6.1 beats/min) and greater reductions in HRV (susceptible, -6.3+/-0.3 vs. resistant, -2.8+/-0.8 ln ms2) in the susceptible dogs compared with the resistant animals. Similar response differences between susceptible and resistant dogs were noted during submaximal exercise. Training significantly reduced the HR and HRV responses to the occlusion (HR, 17.9+/-11.5 beats/min; HRV, -1.2+/-0.8, ln ms2) in the susceptible dogs; similar response reductions were noted during exercise. In contrast, these variables were not altered in the sedentary susceptible dogs. Posttraining, VF could no longer be induced in the susceptible dogs, whereas four sedentary susceptible dogs died during the 10-wk control period, and the remaining seven animals still had VF when tested. Atropine decreased HRV but only induced VF in one of eight trained susceptible dogs. Thus exercise training increased cardiac vagal activity, which was not solely responsible for the training-induced VF protection.

Chronic Omega-3 Polyunsaturated Fatty Acid Treatment Variably Affects Cellular Repolarization in a Healed Post-MI Arrhythmia Model.

INTRODUCTION: Over the last 40 years omega-3 polyunsaturated fatty acids (PUFAs) have been shown to be anti-arrhythmic or pro-arrhythmic depending on the method and duration of administration and model studied. We previously reported that omega-3 PUFAs do not confer anti-arrhythmic properties and are pro-arrhythmic in canine model of sudden cardiac death (SCD). Here, we evaluated the effects of chronic omega-3 PUFA treatment in post-MI animals susceptible (VF+) or resistant (VF-) to ventricular tachyarrhythmias. METHODS: Perforated patch clamp techniques were used to measure cardiomyocyte action potential durations (APD) at 50 and 90% repolarization and short term variability of repolarization. The early repolarizing transient outward potassium current Ito was also studied. RESULTS: Omega-3 PUFAs prolonged the action potential in VF- myocytes at both 50 and 90% repolarization. Short term variability of repolarization was increased in both untreated and treated VF- myocytes vs. CONTROLS: Ito was unaffected by omega-3 PUFA treatment. Omega-3 PUFA treatment attenuated the action potential prolongation in VF+ myocytes, but did not return repolarization to control values. CONCLUSIONS: Omega-3 PUFAs do not confer anti-arrhythmic properties in the setting of healed myocardial infarction in a canine model of SCD. In canines previously resistant to ventricular fibrillation (VF-), omega-3 PUFA treatment prolonged the action potential in VF- myocytes, and may contribute to pro-arrhythmic responses.