Chronic Kidney Disease Induces Proarrhythmic Remodeling.

BACKGROUND: Patients with chronic kidney disease (CKD) are at increased risk of developing cardiac arrhythmogenesis and sudden cardiac death; however, the basis for this association is incompletely known. METHODS: Here, using murine models of CKD, we examined interactions between kidney disease progression and structural, electrophysiological, and molecular cardiac remodeling. RESULTS: C57BL/6 mice with adenine supplemented in their diet developed progressive CKD. Electrocardiographically, CKD mice developed significant QT prolongation and episodes of bradycardia. Optical mapping of isolated-perfused hearts using voltage-sensitive dyes revealed significant prolongation of action potential duration with no change in epicardial conduction velocity. Patch-clamp studies of isolated ventricular cardiomyocytes revealed changes in sodium and potassium currents consistent with action potential duration prolongation. Global transcriptional profiling identified dysregulated expression of cellular stress response proteins RBM3 (RNA-binding motif protein 3) and CIRP (cold-inducible RNA-binding protein) that may underlay the ion channel remodeling. Unexpectedly, we found that female sex is a protective factor in the progression of CKD and its cardiac sequelae. CONCLUSIONS: Our data provide novel insights into the association between CKD and pathologic proarrhythmic cardiac remodeling. Cardiac cellular stress response pathways represent potential targets for pharmacologic intervention for CKD-induced heart rhythm disorders.

Author Correction: Connexin43 expression in bone marrow derived cells contributes to the electrophysiological properties of cardiac scar tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Connexin43 expression in bone marrow derived cells contributes to the electrophysiological properties of cardiac scar tissue.

Cardiac pathologies associated with arrhythmic activity are often accompanied by inflammation. The contribution of inflammatory cells to the electrophysiological properties of injured myocardium is unknown. Myocardial scar cell types and intercellular contacts were analyzed using a three-dimensional reconstruction from serial blockface scanning electron microscopy data. Three distinct cell populations were identified: inflammatory, fibroblastic and endocardial cells. While individual fibroblastic cells interface with a greater number of cells, inflammatory cells have the largest contact area suggesting a role in establishing intercellular electrical connections in scar tissue. Optical mapping was used to study the electrophysiological properties of scars in fetal liver chimeric mice generated using connexin43 knockout donors (bmpKO). Voltage changes were elicited in response to applied current pulses. Isopotential maps showed a steeper pattern of decay with distance from the electrode in scars compared with uninjured regions, suggesting reduced electrical coupling. The tissue decay constant, defined as the distance voltage reaches 37% of the amplitude at the edge of the scar, was 0.48 ± 0.04 mm (n = 11) in the scar of the bmpCTL group and decreased 37.5% in the bmpKO group (n = 10). Together these data demonstrate inflammatory cells significantly contribute to scar electrophysiology through coupling mediated at least partially by connexin43 expression.

Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice.

BACKGROUND: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+]i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. CONCLUSIONS: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Mechanistic investigation of Ca2+ alternans in human heart failure and its modulation by fibroblasts.

BACKGROUND: Heart failure (HF) is characterized, among other factors, by a progressive loss of contractile function and by the formation of an arrhythmogenic substrate, both aspects partially related to intracellular Ca2+ cycling disorders. In failing hearts both electrophysiological and structural remodeling, including fibroblast proliferation, contribute to changes in Ca2+ handling which promote the appearance of Ca2+ alternans (Ca-alt). Ca-alt in turn give rise to repolarization alternans, which promote dispersion of repolarization and contribute to reentrant activity. The computational analysis of the incidence of Ca2+ and/or repolarization alternans under HF conditions in the presence of fibroblasts could provide a better understanding of the mechanisms leading to HF arrhythmias and contractile function disorders. METHODS AND FINDINGS: The goal of the present study was to investigate in silico the mechanisms leading to the formation of Ca-alt in failing human ventricular myocytes and tissues with disperse fibroblast distributions. The contribution of ionic currents variability to alternans formation at the cellular level was analyzed and the results show that in normal ventricular tissue, altered Ca2+ dynamics lead to Ca-alt, which precede APD alternans and can be aggravated by the presence of fibroblasts. Electrophysiological remodeling of failing tissue alone is sufficient to develop alternans. The incidence of alternans is reduced when fibroblasts are present in failing tissue due to significantly depressed Ca2+ transients. The analysis of the underlying ionic mechanisms suggests that Ca-alt are driven by Ca2+-handling protein and Ca2+ cycling dysfunctions in the junctional sarcoplasmic reticulum and that their contribution to alternans occurrence depends on the cardiac remodeling conditions and on myocyte-fibroblast interactions. CONCLUSION: It can thus be concluded that fibroblasts modulate the formation of Ca-alt in human ventricular tissue subjected to heart failure-related electrophysiological remodeling. Pharmacological therapies should thus consider the extent of both the electrophysiological and structural remodeling present in the failing heart.

Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.

Fhf2 gene deletion causes temperature-sensitive cardiac conduction failure.

Fever is a highly conserved systemic response to infection dating back over 600 million years. Although conferring a survival benefit, fever can negatively impact the function of excitable tissues, such as the heart, producing cardiac arrhythmias. Here we show that mice lacking fibroblast growth factor homologous factor 2 (FHF2) have normal cardiac rhythm at baseline, but increasing core body temperature by as little as 3 °C causes coved-type ST elevations and progressive conduction failure that is fully reversible upon return to normothermia. FHF2-deficient cardiomyocytes generate action potentials upon current injection at 25 °C but are unexcitable at 40 °C. The absence of FHF2 accelerates the rate of closed-state and open-state sodium channel inactivation, which synergizes with temperature-dependent enhancement of inactivation rate to severely suppress cardiac sodium currents at elevated temperatures. Our experimental and computational results identify an essential role for FHF2 in dictating myocardial excitability and conduction that safeguards against temperature-sensitive conduction failure.

Connexin43 contributes to electrotonic conduction across scar tissue in the intact heart.

Studies have demonstrated non-myocytes, including fibroblasts, can electrically couple to myocytes in culture. However, evidence demonstrating current can passively spread across scar tissue in the intact heart remains elusive. We hypothesize electrotonic conduction occurs across non-myocyte gaps in the heart and is partly mediated by Connexin43 (Cx43). We investigated whether non-myocytes in ventricular scar tissue are electrically connected to surrounding myocardial tissue in wild type and fibroblast-specific protein-1 driven conditional Cx43 knock-out mice (Cx43fsp1KO). Electrical coupling between the scar and uninjured myocardium was demonstrated by injecting current into the myocardium and recording depolarization in the scar through optical mapping. Coupling was significantly reduced in Cx43fsp1KO hearts. Voltage signals were recorded using microelectrodes from control scars but no signals were obtained from Cx43fsp1KO hearts. Recordings showed significantly decreased amplitude, depolarized resting membrane potential, increased duration and reduced upstroke velocity compared to surrounding myocytes, suggesting that the non-excitable cells in the scar closely follow myocyte action potentials. These results were further validated by mathematical simulations. Optical mapping demonstrated that current delivered within the scar could induce activation of the surrounding myocardium. These data demonstrate non-myocytes in the scar are electrically coupled to myocytes, and coupling depends on Cx43 expression.

A review of the literature on cardiac electrical activity between fibroblasts and myocytes.

Myocardial injuries often lead to fibrotic deposition. This review presents evidence supporting the concept that fibroblasts in the heart electrically couple to myocytes.

Genetically engineered SCN5A mutant pig hearts exhibit conduction defects and arrhythmias.

SCN5A encodes the α subunit of the major cardiac sodium channel Na(V)1.5. Mutations in SCN5A are associated with conduction disease and ventricular fibrillation (VF); however, the mechanisms that link loss of sodium channel function to arrhythmic instability remain unresolved. Here, we generated a large-animal model of a human cardiac sodium channelopathy in pigs, which have cardiac structure and function similar to humans, to better define the arrhythmic substrate. We introduced a nonsense mutation originally identified in a child with Brugada syndrome into the orthologous position (E558X) in the pig SCN5A gene. SCN5A(E558X/+) pigs exhibited conduction abnormalities in the absence of cardiac structural defects. Sudden cardiac death was not observed in young pigs; however, Langendorff-perfused SCN5A(E558X/+) hearts had an increased propensity for pacing-induced or spontaneous VF initiated by short-coupled ventricular premature beats. Optical mapping during VF showed that activity often began as an organized focal source or broad wavefront on the right ventricular (RV) free wall. Together, the results from this study demonstrate that the SCN5A(E558X/+) pig model accurately phenocopies many aspects of human cardiac sodium channelopathy, including conduction slowing and increased susceptibility to ventricular arrhythmias.

Fibroblast KATP currents modulate myocyte electrophysiology in infarcted hearts.

Cardiac metabolism remains altered for an extended period of time after myocardial infarction. Studies have shown fibroblasts from normal hearts express KATP channels in culture. It is unknown whether fibroblasts from infarcted hearts express KATP channels and whether these channels contribute to scar and border zone electrophysiology. KATP channel subunit expression levels were determined in fibroblasts isolated from normal hearts (Fb), and scar (sMI-Fb) and remote (rMI-Fb) regions of left anterior descending coronary artery (LAD) ligated rat hearts. Whole cell KATP current density was determined with patch clamp. Action potential duration (APD) was measured with optical mapping in myocyte-only cultures and heterocellular cultures with fibroblasts with and without 100 µmol/l pinacidil. Whole heart optical mapping was used to assess KATP channel activity following LAD ligation. Pinacidil activated a potassium current (35.4 ± 7.5 pA/pF at 50 mV) in sMI-Fb that was inhibited with 10 µmol/l glibenclamide. Kir6.2 and SUR2 transcript levels were elevated in sMI-Fb. Treatment with Kir6.2 short interfering RNA decreased KATP currents (87%) in sMI-Fb. Treatment with pinacidil decreased APD (26%) in co-cultures with sMI-Fb. APD values were prolonged in LAD ligated hearts after perfusion with glibenclamide. KATP channels are present in fibroblasts from the scar and border zones of infarcted hearts. Activation of fibroblast KATP channels could modulate the electrophysiological substrate beyond the acute ischemic event. Targeting fibroblast KATP channels could represent a novel therapeutic approach to modify border zone electrophysiology after cardiac injury.

The origin and arrhythmogenic potential of fibroblasts in cardiac disease.

Fibroblasts play a major role in normal cardiac physiology and in the response of the heart to injury and disease. Cardiac electrophysiological research has primarily focused on the mechanisms of remodeling that accompany cardiac disease with an emphasis on myocyte electrophysiology. Recently, there has been increasing interest in the potential role of fibroblasts in cardiac electrophysiology. This review focuses on the arrhythmia mechanisms involving interactions between myocytes and fibroblasts. We also discuss the available evidence supporting the contribution of intracardiac and extracardiac sources to the fibroblast and myofibroblast populations in diseased hearts.

Spinal cord stimulation protects against atrial fibrillation induced by tachypacing.

BACKGROUND: Spinal cord stimulation (SCS) has been shown to modulate atrial electrophysiology and confer protection against ischemia and ventricular arrhythmias in animal models. OBJECTIVE: To determine whether SCS reduces the susceptibility to atrial fibrillation (AF) induced by tachypacing (TP). METHODS: In 21 canines, upper thoracic SCS systems and custom cardiac pacing systems were implanted. Right atrial and left atrial effective refractory periods were measured at baseline and after 15 minutes of SCS. Following recovery in a subset of canines, pacemakers were turned on to induce AF by alternately delivering TP and searching for AF. Canines were randomized to no SCS therapy (CTL) or intermittent SCS therapy on the initiation of TP (EARLY) or after 8 weeks of TP (LATE). AF burden (percent AF relative to total sense time) and AF inducibility (percentage of TP periods resulting in AF) were monitored weekly. After 15 weeks, echocardiography and histology were performed. RESULTS: Effective refractory periods increased by 21 ± 14 ms (P = .001) in the left atrium and 29 ± 12 ms (P = .002) in the right atrium after acute SCS. AF burden was reduced for 11 weeks in EARLY compared with CTL (P <.05) animals. AF inducibility remained lower by week 15 in EARLY compared with CTL animals (32% ± 10% vs 91% ± 6%; P <.05). AF burden and inducibility were not significantly different between LATE and CTL animals. There were no structural differences among any groups. CONCLUSIONS: SCS prolonged atrial effective refractory periods and reduced AF burden and inducibility in a canine AF model induced by TP. These data suggest that SCS may represent a treatment option for AF.

Mice with cardiac overexpression of peroxisome proliferator-activated receptor γ have impaired repolarization and spontaneous fatal ventricular arrhythmias.

BACKGROUND: Diabetes mellitus and obesity, which confer an increased risk of sudden cardiac death, are associated with cardiomyocyte lipid accumulation and altered cardiac electric properties, manifested by prolongation of the QRS duration and QT interval. It is difficult to distinguish the contribution of cardiomyocyte lipid accumulation from the contribution of global metabolic defects to the increased incidence of sudden death and electric abnormalities. METHODS AND RESULTS: In order to study the effects of metabolic abnormalities on arrhythmias without the complex systemic effects of diabetes mellitus and obesity, we studied transgenic mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor γ 1 (PPARγ1) via the cardiac α-myosin heavy-chain promoter. The PPARγ transgenic mice develop abnormal accumulation of intracellular lipids and die as young adults before any significant reduction in systolic function. Using implantable ECG telemeters, we found that these mice have prolongation of the QRS and QT intervals and spontaneous ventricular arrhythmias, including polymorphic ventricular tachycardia and ventricular fibrillation. Isolated cardiomyocytes demonstrated prolonged action potential duration caused by reduced expression and function of the potassium channels responsible for repolarization. Short-term exposure to pioglitazone, a PPARγ agonist, had no effect on mortality or rhythm in WT mice but further exacerbated the arrhythmic phenotype and increased the mortality in the PPARγ transgenic mice. CONCLUSIONS: Our findings support an important link between PPARγ activation, cardiomyocyte lipid accumulation, ion channel remodeling, and increased cardiac mortality.

Unique properties of the ATP-sensitive K⁺ channel in the mouse ventricular cardiac conduction system.

Background- The specialized cardiac conduction system (CCS) expresses a unique complement of ion channels that confer a specific electrophysiological profile. ATP-sensitive potassium (K(ATP)) channels in these myocytes have not been systemically investigated. Methods and Results- We recorded K(ATP) channels in isolated CCS myocytes using Cntn2-EGFP reporter mice. The CCS K(ATP) channels were less sensitive to inhibitory cytosolic ATP compared with ventricular channels and more strongly activated by MgADP. They also had a smaller slope conductance. The 2 types of channels had similar intraburst open and closed times, but the CCS K(ATP) channel had a prolonged interburst closed time. CCS K(ATP) channels were strongly activated by diazoxide and less by levcromakalim, whereas the ventricular K(ATP) channel had a reverse pharmacological profile. CCS myocytes express elevated levels of Kir6.1 but reduced Kir6.2 and SUR2A mRNA compared with ventricular myocytes (SUR1 expression was negligible). SUR2B mRNA expression was higher in CCS myocytes relative to SUR2A. Canine Purkinje fibers expressed higher levels of Kir6.1 and SUR2B protein relative to the ventricle. Numeric simulation predicts a high sensitivity of the Purkinje action potential to changes in ATP:ADP ratio. Cardiac conduction time was prolonged by low-flow ischemia in isolated, perfused mouse hearts, which was prevented by glibenclamide. Conclusions- These data imply a differential electrophysiological response (and possible contribution to arrhythmias) of the ventricular CCS to K(ATP) channel opening during periods of ischemia.

Remodeling of atrial ATP-sensitive K⁺ channels in a model of salt-induced elevated blood pressure.

Hypertension is associated with the development of atrial fibrillation; however, the electrophysiological consequences of this condition remain poorly understood. ATP-sensitive K(+) (K(ATP)) channels, which contribute to ventricular arrhythmias, are also expressed in the atria. We hypothesized that salt-induced elevated blood pressure (BP) leads to atrial K(ATP) channel activation and increased arrhythmia inducibility. Elevated BP was induced in mice with a high-salt diet (HS) for 4 wk. High-resolution optical mapping was used to measure atrial arrhythmia inducibility, effective refractory period (ERP), and action potential duration at 90% repolarization (APD(90)). Excised patch clamping was performed to quantify K(ATP) channel properties and density. K(ATP) channel protein expression was also evaluated. Atrial arrhythmia inducibility was 22% higher in HS hearts compared with control hearts. ERP and APD(90) were significantly shorter in the right atrial appendage and left atrial appendage of HS hearts compared with control hearts. Perfusion with 1 µM glibenclamide or 300 µM tolbutamide significantly decreased arrhythmia inducibility and prolonged APD(90) in HS hearts compared with untreated HS hearts. K(ATP) channel density was 156% higher in myocytes isolated from HS animals compared with control animals. Sulfonylurea receptor 1 protein expression was increased in the left atrial appendage and right atrial appendage of HS animals (415% and 372% of NS animals, respectively). In conclusion, K(ATP) channel activation provides a mechanistic link between salt-induced elevated BP and increased atrial arrhythmia inducibility. The findings of this study have important implications for the treatment and prevention of atrial arrhythmias in the setting of hypertensive heart disease and may lead to new therapeutic approaches.

Spatiotemporal electrophysiological changes in a murine ablation model.

AIMS: High recurrence rates after complex radiofrequency ablation procedures, such as for atrial fibrillation, remain a major clinical problem. Local electrophysiological changes that occur following cardiac ablation therapy are incompletely described in the literature. The purpose of this study was to determine whether alterations in conduction velocity, action potential duration (APD), and effective refractory period resolve dynamically following cardiac ablation. METHODS AND RESULTS: Lesions were delivered to the right ventricle of mice using a subxiphoid approach. The sham-operated control group (SHAM) received the same procedure without energy delivery. Hearts were isolated at 0, 1, 7, 30, and 60 days following the procedure and electrophysiological parameters were obtained using high-resolution optical mapping with a voltage-sensitive dye. Conduction velocity was significantly decreased at the lesion border in the 0, 7, and 30 day groups compared to SHAM. APD(70) at the lesion border was significantly increased at all time points compared to SHAM. Effective refractory period was significantly increased at the lesion border at 0, 1, 7, and 30 days but not at 60 days post-ablation. This study demonstrated that post-ablation electrophysiological changes take place immediately following energy delivery and resolve within 60 days. CONCLUSIONS: Cardiac ablation causes significant electrophysiological changes both within the lesion and beyond the border zone. Late recovery of electrical conduction in individual lesions is consistent with clinical data demonstrating that arrhythmia recurrence is associated with failure to maintain bi-directional conduction block.