A comprehensive protocol combining in vivo and ex vivo electrophysiological experiments in an arrhythmogenic animal model.

Ventricular arrhythmias contribute significantly to cardiovascular mortality, with coronary artery disease as the predominant underlying cause. Understanding the mechanisms of arrhythmogenesis is essential to identify proarrhythmic factors and develop novel approaches for antiarrhythmic prophylaxis and treatment. Animal models are vital in basic research on cardiac arrhythmias, encompassing molecular, cellular, ex vivo whole heart, and in vivo models. Most studies use either in vivo protocols lacking important information on clinical relevance or exclusively ex vivo protocols, thereby missing the opportunity to explore underlying mechanisms. Consequently, interpretation may be difficult due to dissimilarities in animal models, interventions, and individual properties across animals. Moreover, proarrhythmic effects observed in vivo are often not replicated in corresponding ex vivo preparations during mechanistic studies. We have established a protocol to perform both an in vivo and ex vivo electrophysiological characterization in an arrhythmogenic rat model with heart failure following myocardial infarction. The same animal is followed throughout the experiment. In vivo methods involve intracardiac programmed electrical stimulation and external defibrillation to terminate sustained ventricular arrhythmia. Ex vivo methods conducted on the Langendorff-perfused heart include an electrophysiological study with optical mapping of regional action potentials, conduction velocities, and dispersion of electrophysiological properties. By exploring the retention of the in vivo proarrhythmic phenotype ex vivo, we aim to examine whether the subsequent ex vivo detailed measurements are relevant to in vivo pathological behavior. This protocol can enhance greater understanding of cardiac arrhythmias by providing a standardized, yet adaptable model for evaluating arrhythmogenicity or antiarrhythmic interventions in cardiac diseases.NEW & NOTEWORTHY Rodent models are widely used in arrhythmia research. However, most studies do not standardize clinically relevant in vivo and ex vivo techniques to support their conclusions. Here, we present a comprehensive electrophysiological protocol in an arrhythmogenic rat model, connecting in vivo and ex vivo programmed electrical stimulation with optical mapping. By establishing this protocol, we aim to facilitate the adoption of a standardized model for investigating arrhythmias, enhancing research rigor and comparability in this field.

Cardiomyocyte ryanodine receptor clusters expand and coalesce after application of isoproterenol.

Earlier work has shown that ventricular ryanodine receptors (RyR2) within a cluster rearrange on phosphorylation as well as with a number of other stimuli. Using dSTORM, we investigated the effects of 300 nmol/liter isoproterenol on RyR2 clusters. In rat ventricular cardiomyocytes, there was a symmetrical enlargement of RyR2 cluster areas, a decrease in the edge-to-edge nearest neighbor distance, and distribution changes that suggested movement to increase the cluster areas by coalescence. The surface area covered by the phosphorylated clusters was significantly greater than in the control cells, as was the cluster density. This latter change was accompanied by a decreased cluster fragmentation, implying that new tetramers were brought into the sarcoplasmic reticulum. We propose a possible mechanism to explain these changes. We also visualized individual RyR2 tetramers and confirmed our earlier electron-tomographic finding that the tetramers are in a disorganized but non-random array occupying about half of the cluster area. Multiclusters, cluster groups defined by the maximum distance between their members, were analyzed for various distances. At 100 nm, the areas occupied by the multiclusters just exceeded those of the single clusters, and more than half of the multiclusters had only a single subcluster that could initiate a spark. Phosphorylation increased the size of the multiclusters, markedly so for distances >100 nm. There was no relationship between the number of subclusters in a group and the area covered by it. We conclude that isoproterenol induces rapid, significant, changes in the molecular architecture of excitation-contraction coupling.

A circadian clock in the sinus node mediates day-night rhythms in Hcn4 and heart rate.

BACKGROUND: Heart rate follows a diurnal variation, and slow heart rhythms occur primarily at night. OBJECTIVE: The lower heart rate during sleep is assumed to be neural in origin, but here we tested whether a day-night difference in intrinsic pacemaking is involved. METHODS: In vivo and in vitro electrocardiographic recordings, vagotomy, transgenics, quantitative polymerase chain reaction, Western blotting, immunohistochemistry, patch clamp, reporter bioluminescence recordings, and chromatin immunoprecipitation were used. RESULTS: The day-night difference in the average heart rate of mice was independent of fluctuations in average locomotor activity and persisted under pharmacological, surgical, and transgenic interruption of autonomic input to the heart. Spontaneous beating rate of isolated (ie, denervated) sinus node (SN) preparations exhibited a day-night rhythm concomitant with rhythmic messenger RNA expression of ion channels including hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4). In vitro studies demonstrated 24-hour rhythms in the human HCN4 promoter and the corresponding funny current. The day-night heart rate difference in mice was abolished by HCN block, both in vivo and in the isolated SN. Rhythmic expression of canonical circadian clock transcription factors, for example, Brain and muscle ARNT-Like 1 (BMAL1) and Cryptochrome (CRY) was identified in the SN and disruption of the local clock (by cardiomyocyte-specific knockout of Bmal1) abolished the day-night difference in Hcn4 and intrinsic heart rate. Chromatin immunoprecipitation revealed specific BMAL1 binding sites on Hcn4, linking the local clock with intrinsic rate control. CONCLUSION: The circadian variation in heart rate involves SN local clock-dependent Hcn4 rhythmicity. Data reveal a novel regulator of heart rate and mechanistic insight into bradycardia during sleep.

Exercise training reveals micro-RNAs associated with improved cardiac function and electrophysiology in rats with heart failure after myocardial infarction.

AIMS: Endurance training improves aerobic fitness and cardiac function in individuals with heart failure. However, the underlying mechanisms are not well characterized. Exercise training could therefore act as a tool to discover novel targets for heart failure treatment. We aimed to associate changes in Ca2+ handling and electrophysiology with micro-RNA (miRNA) profile in exercise trained heart failure rats to establish which miRNAs induce heart failure-like effects in Ca2+ handling and electrophysiology. METHODS AND RESULTS: Post-myocardial infarction (MI) heart failure was induced in Sprague Dawley rats. Rats with MI were randomized to sedentary control (sed), moderate (mod)- or high-intensity (high) endurance training for 8 weeks. Exercise training improved cardiac function, Ca2+ handling and electrophysiology including reduced susceptibility to arrhythmia in an exercise intensity-dependent manner where high intensity gave a larger effect. Fifty-five miRNAs were significantly regulated (up or down) in MI-sed, of which 18 and 3 were changed towards Sham-sed in MI-high and MI-mod, respectively. Thereafter we experimentally altered expression of these "exercise-miRNAs" individually in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM) in the same direction as they were changed in MI. Of the "exercise-miRNAs", miR-214-3p prolonged AP duration, whereas miR-140 and miR-208a shortened AP duration. miR-497-5p prolonged Ca2+ release whereas miR-214-3p and miR-31a-5p prolonged Ca2+ decay. CONCLUSION: Using exercise training as a tool, we discovered that miR-214-3p, miR-497-5p, miR-31a-5p contribute to heart-failure like behaviour in Ca2+ handling and electrophysiology and could be potential treatment targets.

Targeting miR-423-5p Reverses Exercise Training-Induced HCN4 Channel Remodeling and Sinus Bradycardia.

RATIONALE: Downregulation of the pacemaking ion channel, HCN4 (hyperpolarization-activated cyclic nucleotide gated channel 4), and the corresponding ionic current, If, underlies exercise training-induced sinus bradycardia in rodents. If this occurs in humans, it could explain the increased incidence of bradyarrhythmias in veteran athletes, and it will be important to understand the underlying processes. OBJECTIVE: To test the role of HCN4 in the training-induced bradycardia in human athletes and investigate the role of microRNAs (miRs) in the repression of HCN4. METHODS AND RESULTS: As in rodents, the intrinsic heart rate was significantly lower in human athletes than in nonathletes, and in all subjects, the rate-lowering effect of the HCN selective blocker, ivabradine, was significantly correlated with the intrinsic heart rate, consistent with HCN repression in athletes. Next-generation sequencing and quantitative real-time reverse transcription polymerase chain reaction showed remodeling of miRs in the sinus node of swim-trained mice. Computational predictions highlighted a prominent role for miR-423-5p. Interaction between miR-423-5p and HCN4 was confirmed by a dose-dependent reduction in HCN4 3'-untranslated region luciferase reporter activity on cotransfection with precursor miR-423-5p (abolished by mutation of predicted recognition elements). Knockdown of miR-423-5p with anti-miR-423-5p reversed training-induced bradycardia via rescue of HCN4 and If. Further experiments showed that in the sinus node of swim-trained mice, upregulation of miR-423-5p (intronic miR) and its host gene, NSRP1, is driven by an upregulation of the transcription factor Nkx2.5. CONCLUSIONS: HCN remodeling likely occurs in human athletes, as well as in rodent models. miR-423-5p contributes to training-induced bradycardia by targeting HCN4. This work presents the first evidence of miR control of HCN4 and heart rate. miR-423-5p could be a therapeutic target for pathological sinus node dysfunction in veteran athletes.

Biophysical characterization of the underappreciated and important relationship between heart rate variability and heart rate.

Heart rate (HR) variability (HRV; beat-to-beat changes in the R-wave to R-wave interval) has attracted considerable attention during the past 30+ years (PubMed currently lists >17 000 publications). Clinically, a decrease in HRV is correlated to higher morbidity and mortality in diverse conditions, from heart disease to fetal distress. It is usually attributed to fluctuation in cardiac autonomic nerve activity. We calculated HRV parameters from a variety of cardiac preparations (including humans, living animals, Langendorff-perfused heart, and single sinoatrial nodal cell) in diverse species, combining this with data from previously published articles. We show that regardless of conditions, there is a universal exponential decay-like relationship between HRV and HR. Using 2 biophysical models, we develop a theory for this and confirm that HRV is primarily dependent on HR and cannot be used in any simple way to assess autonomic nerve activity to the heart. We suggest that the correlation between a change in HRV and altered morbidity and mortality is substantially attributable to the concurrent change in HR. This calls for re-evaluation of the findings from many articles that have not adjusted properly or at all for HR differences when comparing HRV in multiple circumstances.

Comparison of left versus right atrial myocardium in patients with sinus rhythm or atrial fibrillation - an assessment of mitochondrial function and microRNA expression.

Several of the cellular alterations involved in atrial fibrillation (AF) may be linked to mitochondrial function and altered microRNA (miR) expression. A majority of studies on human myocardium involve right atrial (RA) tissue only. There are indications that AF may affect the two atria differentially. This study aimed to compare interatrial differences in mitochondrial respiration and miR expression in the RA versus left atrium (LA) within patients with sinus rhythm (SR) and AF. Thirty-seven patients with AF (n = 21) or SR (n = 16), undergoing coronary artery bypass surgery and/or heart valve surgery, were included. Myocardial biopsies were obtained from RA and LA appendages. Mitochondrial respiration was assessed in situ in permeabilized myocardium. MiR array and real-time quantitative polymerase chain reaction were performed to evaluate miR expression. Mitochondrial respiratory rates were similar in RA versus LA. Expression of miR-100, -10b, -133a, -133b, -146a, -155, -199a-5p, -208b, and -30b were different between the atria in both SR and AF patients. In contrast, differential expression was observed between RA versus LA for miR-93 in patients with SR only, and for miR-1, -125b, -142-5p, -208a, and -92b within AF patients only. These results indicate that mitochondrial respiratory capacity is similar in the RA and LA of patients with SR and AF. Differences in miR expressional profiles are observed between the RA versus LA in both SR and AF, and several interatrial differences in miR expression diverge between SR and AF. These findings may contribute to the understanding of how AF pathophysiology may affect the two atria differently.

Exercise training reduces resting heart rate via downregulation of the funny channel HCN4.

Endurance athletes exhibit sinus bradycardia, that is a slow resting heart rate, associated with a higher incidence of sinus node (pacemaker) disease and electronic pacemaker implantation. Here we show that training-induced bradycardia is not a consequence of changes in the activity of the autonomic nervous system but is caused by intrinsic electrophysiological changes in the sinus node. We demonstrate that training-induced bradycardia persists after blockade of the autonomous nervous system in vivo in mice and in vitro in the denervated sinus node. We also show that a widespread remodelling of pacemaker ion channels, notably a downregulation of HCN4 and the corresponding ionic current, If. Block of If abolishes the difference in heart rate between trained and sedentary animals in vivo and in vitro. We further observe training-induced downregulation of Tbx3 and upregulation of NRSF and miR-1 (transcriptional regulators) that explains the downregulation of HCN4. Our findings provide a molecular explanation for the potentially pathological heart rate adaptation to exercise training.

Mitochondrial respiration and microRNA expression in right and left atrium of patients with atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia with a potential to cause serious complications. Mitochondria play central roles in cardiomyocyte function and have been implicated in AF pathophysiology. MicroRNA (miR) are suggested to influence both mitochondrial function and the development of AF. Yet mitochondrial function and miR expression remain largely unexplored in human atrial tissue. This study aims to investigate mitochondrial function and miR expression in the right (RA) and left atria (LA) of patients with AF and sinus rhythm (SR). Myocardial tissue from the RA and LA appendages was investigated in 37 patients with AF (n = 21) or SR (n = 16) undergoing coronary artery bypass surgery and/or heart valve surgery. Mitochondrial respiration was measured in situ after tissue permeabilization by saponin. MiR expression was assessed by miR array and real-time quantitative reverse-transcription polymerase chain reaction. Maximal mitochondrial respiratory rate was increased in both RA and LA tissue of patients with AF vs. SR. Biatrial downregulation of miR-208a and upregulation of miR-106b, -144, and -451 were observed in AF vs. SR. In addition, miR-15b was upregulated in AF within RA only, and miR-106a, -18a, -18b, -19a, -19b, -23a, -25, -30a, -363, -486-5p, -590-5p, and -93 were upregulated in AF within LA only. These findings suggest that mitochondrial function and miR are involved in AF pathophysiology and should be areas of focus in the exploration for potential novel therapeutic targets.

Atrial myocyte function and Ca2+ handling is associated with inborn aerobic capacity.

BACKGROUND: Although high aerobic capacity is associated with effective cardiac function, the effect of aerobic capacity on atrial function, especially in terms of cellular mechanisms, is not known. We aimed to investigate whether rats with low inborn maximal oxygen uptake (VO2 max) had impaired atrial myocyte contractile function when compared to rats with high inborn VO2 max. METHODS AND RESULTS: Atrial myocyte function was depressed in Low Capacity Runners (LCR) relative to High Capacity Runners (HCR) which was associated with impaired Ca(2+) handling. Fractional shortening was 52% lower at 2 Hz and 60% lower at 5 Hz stimulation while time to 50% relengthening was 43% prolonged and 55% prolonged, respectively. Differences in Ca(2+) amplitude and diastolic Ca(2+) level were observed at 5 Hz stimulation where Ca(2+) amplitude was 70% lower and diastolic Ca(2+) level was 11% higher in LCR rats. Prolonged time to 50% Ca(2+) decay was associated with reduced sarcoplasmic reticulum (SR) Ca(2+) ATPase function in LCR (39%). Na(+)/Ca(2+) exchanger activity was comparable between the groups. Diastolic SR Ca(2+) leak was increased by 109%. This could be partly explained by increased ryanodine receptors phosphorylation at the Ca(2+)-calmodulin-dependent protein kinase-II specific Ser-2814 site in LCR rats. T-tubules were present in 68% of HCR cells whereas only 33% LCR cells had these structures. In HCR, the significantly higher numbers of cells with T-tubules were combined with greater numbers of myocytes where Ca(2+) release in the cell occurred simultaneously in central and peripheral regions, giving rise to faster and more spatial homogenous Ca(2+)-signal onset. CONCLUSION: This data demonstrates that contrasting for low or high aerobic capacity leads to diverse functional and structural remodelling of atrial myocytes, with impaired contractile function in LCR compared to HCR rats.

Aerobic interval training partly reverse contractile dysfunction and impaired Ca2+ handling in atrial myocytes from rats with post infarction heart failure.

BACKGROUND: There is limited knowledge about atrial myocyte Ca(2+) handling in the failing hearts. The aim of this study was to examine atrial myocyte contractile function and Ca(2+) handling in rats with post-infarction heart failure (HF) and to examine whether aerobic interval training could reverse a potential dysfunction. METHODS AND RESULTS: Post-infarction HF was induced in Sprague Dawley rats by ligation of the left descending coronary artery. Atrial myocyte shortening was depressed (p<0.01) and time to relaxation was prolonged (p<0.01) in sedentary HF-rats compared to healthy controls. This was associated with decreased Ca(2+) amplitude, decreased SR Ca(2+) content, and slower Ca(2+) transient decay. Atrial myocytes from HF-rats had reduced sarcoplasmic reticulum Ca(2+) ATPase activity, increased Na(+)/Ca(2+)-exchanger activity and increased diastolic Ca(2+) leak through ryanodine receptors. High intensity aerobic interval training in HF-rats restored atrial myocyte contractile function and reversed changes in atrial Ca(2+) handling in HF. CONCLUSION: Post infarction HF in rats causes profound impairment in atrial myocyte contractile function and Ca(2+) handling. The observed dysfunction in atrial myocytes was partly reversed after aerobic interval training.