Vadim V. Fedorov

Transmural dispersion of repolarization in failing and nonfailing human ventricle.

Rationale: Transmural dispersion of repolarization has been shown to play a role in the genesis of ventricular tachycardia and fibrillation in different animal models of heart failure (HF). Heterogeneous changes of repolarization within the midmyocardial population of ventricular cells have been considered an important contributor to the HF phenotype. However, there is limited electrophysiological data from the human heart. Objective: To study electrophysiological remodeling of transmural repolarization in the failing and nonfailing human hearts. Methods and Results: We optically mapped the action potential duration (APD) in the coronary-perfused scar-free posterior-lateral left ventricular free wall wedge preparations from failing (n=5) and nonfailing (n=5) human hearts. During slow pacing (S1S1=2000 ms), in the nonfailing hearts we observed significant transmural APD gradient: subepicardial, midmyocardial, and subendocardial APD80 were 383±21, 455±20, and 494±22 ms, respectively. In 60% of nonfailing hearts (3 of 5), we found midmyocardial islands of cells that presented a distinctly long APD (537±40 ms) and a steep local APD gradient (27±7 ms/mm) compared with the neighboring myocardium. HF resulted in prolongation of APD80: 477±22 ms, 495±29 ms, and 506±35 ms for the subepi-, mid-, and subendocardium, respectively, while reducing transmural APD80 difference from 111±13 to 29±6 ms (P<0.005) and presence of any prominent local APD gradient. In HF, immunostaining revealed a significant reduction of connexin43 expression on the subepicardium. Conclusions: We present for the first time direct experimental evidence of a transmural APD gradient in the human heart. HF results in the heterogeneous prolongation of APD, which significantly reduces the transmural and local APD gradients.

Transmural Heterogeneity and Remodeling of Ventricular Excitation-Contraction Coupling in Human Heart Failure

Background— Excitation-contraction (EC) coupling is altered in end-stage heart failure. However, spatial heterogeneity of this remodeling has not been established at the tissue level in failing human heart. The objective of this article was to study functional remodeling of excitation-contraction coupling and calcium handling in failing and nonfailing human hearts. Methods and Results— We simultaneously optically mapped action potentials and calcium transients in coronary perfused left ventricular wedge preparations from nonfailing (n=6) and failing (n=5) human hearts. Our major findings are the following. First, calcium transient duration minus action potential duration was longer at subendocardium in failing compared with nonfailing hearts during bradycardia (40 bpm). Second, the transmural gradient of calcium transient duration was significantly smaller in failing hearts compared with nonfailing hearts at fast pacing rates (100 bpm). Third, calcium transient in failing hearts had a flattened plateau at the midmyocardium and exhibited a 2-component slow rise at the subendocardium in 3 failing hearts. Fourth, calcium transient relaxation was slower at the subendocardium than at the subepicardium in both groups. Protein expression of sarcoplasmic reticulum Ca2+-ATPase 2a was lower at the subendocardium than the subepicardium in both nonfailing and failing hearts. Sarcoplasmic reticulum Ca2+-ATPase 2a protein expression at subendocardium was lower in hearts with ischemic cardiomyopathy compared with those with nonischemic cardiomyopathy. Conclusions— For the first time, we present direct experimental evidence of transmural heterogeneity of excitation-contraction coupling and calcium handling in human hearts. End-stage heart failure is associated with the heterogeneous remodeling of excitation-contraction coupling and calcium handling.

Optical Mapping of the Isolated Coronary-Perfused Human Sinus Node

OBJECTIVES We sought to confirm our hypothesis that the human sinoatrial node (SAN) is functionally insulated from the surrounding atrial myocardium except for several exit pathways that electrically bridge the nodal tissue and atrial myocardium. BACKGROUND The site of origin and pattern of excitation within the human SAN has not been directly mapped. METHODS The SAN was optically mapped in coronary-perfused preparations from nonfailing human hearts (n = 4, age 54 ± 15 years) using the dye Di-4-ANBDQBS and blebbistatin. The SAN 3-dimensional structure was reconstructed using histology. RESULTS Optical recordings from the SAN had diastolic depolarization and multiple upstroke components, which corresponded to the separate excitations of the SAN and atrial layers. Excitation originated in the middle of the SAN (66 ± 17 beats/min), and then spread slowly (1 to 18 cm/s) and anisotropically. After a 82 ± 17 ms conduction delay within the SAN, the atrial myocardium was excited via superior, middle, and/or inferior sinoatrial conduction pathways. Atrial excitation was initiated 9.4 ± 4.2 mm from the leading pacemaker site. The oval 14.3 ± 1.5 mm × 6.7 ± 1.6 mm × 1.0 ± 0.2 mm SAN structure was functionally insulated from the atrium by connective tissue, fat, and coronary arteries, except for these pathways. CONCLUSIONS These data demonstrated for the first time, to our knowledge, the location of the leading SAN pacemaker site, the pattern of excitation within the human SAN, and the conduction pathways into the right atrium. The existence of these pathways explains why, even during normal sinus rhythm, atrial breakthroughs could arise from a region parallel to the crista terminalis that is significantly larger (26.1 ± 7.9 mm) than the area of the anatomically defined SAN.

Conduction Remodeling in Human End-Stage Nonischemic Left Ventricular Cardiomyopathy

Background— Several arrhythmogenic mechanisms have been inferred from animal heart failure models. However, the translation of these hypotheses is difficult because of the lack of functional human data. We aimed to investigate the electrophysiological substrate for arrhythmia in human end-stage nonischemic cardiomyopathy. Methods and Results— We optically mapped the coronary-perfused left ventricular wedge preparations from human hearts with end-stage nonischemic cardiomyopathy (heart failure, n=10) and nonfailing hearts (NF, n=10). Molecular remodeling was studied with immunostaining, Western blotting, and histological analyses. Heart failure produced heterogeneous prolongation of action potential duration resulting in the decrease of transmural action potential duration dispersion (64±12 ms versus 129±15 ms in NF, P<0.005). In the failing hearts, transmural activation was significantly slowed from the endocardium (39±3 cm/s versus 49±2 cm/s in NF, P=0.008) to the epicardium (28±3 cm/s versus 40±2 cm/s in NF, P=0.008). Conduction slowing was likely due to connexin 43 (Cx43) downregulation, decreased colocalization of Cx43 with N-cadherin (40±2% versus 52±5% in NF, P=0.02), and an altered distribution of phosphorylated Cx43 isoforms by the upregulation of the dephosphorylated Cx43 in both the subendocardium and subepicardium layers. Failing hearts further demonstrated spatially discordant conduction velocity alternans which resulted in nonuniform propagation discontinuities and wave breaks conditioned by strands of increased interstitial fibrosis (fibrous tissue content in heart failure 16.4±7.7 versus 9.9±1.4% in NF, P=0.02). Conclusions— Conduction disorder resulting from the anisotropic downregulation of Cx43 expression, the reduction of Cx43 phosphorylation, and increased fibrosis is likely to be a critical component of arrhythmogenic substrate in patients with nonischemic cardiomyopathy.

Structural and Functional Evidence for Discrete Exit Pathways That Connect the Canine Sinoatrial Node and Atria

Surface electrode recordings cannot delineate the activation within the human or canine sinoatrial node (SAN) because they are intramural structures. Thus, the site of origin of excitation and conduction pathway(s) within the SAN of these mammals remains unknown. Canine right atrial preparations (n=7) were optically mapped. The SAN 3D structure and protein expression were mapped using immunohistochemistry. SAN optical action potentials had diastolic depolarization and multiple upstroke components that corresponded to the separate excitations of the node and surface atrial layers. Pacing-induced SAN exit block eliminated atrial optical action potential components but retained SAN optical action potential components. Excitation originated in the SAN (cycle length, 557±72 ms) and slowly spread (1.2 to 14 cm/sec) within the SAN, failing to directly excite the crista terminalis and intraatrial septum. After a 49±22 ms conduction delay within the SAN, excitation reached the atrial myocardium via superior and/or inferior sinoatrial exit pathways 8.8±3.2 mm from the leading pacemaker site. The ellipsoidal 13.7±2.8/4.9±0.6 mm SAN structure was functionally insulated from the atrium. This insulation coincided with connexin43-negative regions at the borders of the node, connective tissue, and coronary arteries. During normal sinus rhythm, the canine SAN is functionally insulated from the surrounding atrial myocardium except for 2 (or more) narrow superior and inferior sinoatrial exit pathways separated by 12.8±4.1 mm. Conduction failure in these sinoatrial exit pathways leads to SAN exit block and is a modulator of heart rate.

Effects of KATP channel openers diazoxide and pinacidil in coronary-perfused atria and ventricles from failing and non-failing human hearts

This study compared the effects of ATP-regulated potassium channel (K(ATP)) openers, diazoxide and pinacidil, on diseased and normal human atria and ventricles. We optically mapped the endocardium of coronary-perfused right (n=11) or left (n=2) posterior atrial-ventricular free wall preparations from human hearts with congestive heart failure (CHF, n=8) and non-failing human hearts without (NF, n=3) or with (INF, n=2) infarction. We also analyzed the mRNA expression of the K(ATP) targets K(ir)6.1, K(ir)6.2, SUR1, and SUR2 in the left atria and ventricles of NF (n=8) and CHF (n=4) hearts. In both CHF and INF hearts, diazoxide significantly decreased action potential durations (APDs) in atria (by -21±3% and -27±13%, p<0.01) and ventricles (by -28±7% and -28±4%, p<0.01). Diazoxide did not change APD (0±5%) in NF atria. Pinacidil significantly decreased APDs in both atria (-46 to -80%, p<0.01) and ventricles (-65 to -93%, p<0.01) in all hearts studied. The effect of pinacidil on APD was significantly higher than that of diazoxide in both atria and ventricles of all groups (p<0.05). During pinacidil perfusion, burst pacing induced flutter/fibrillation in all atrial and ventricular preparations with dominant frequencies of 14.4±6.1 Hz and 17.5±5.1 Hz, respectively. Glibenclamide (10 μM) terminated these arrhythmias and restored APDs to control values. Relative mRNA expression levels of K(ATP) targets were correlated to functional observations. Remodeling in response to CHF and/or previous infarct potentiated diazoxide-induced APD shortening. The activation of atrial and ventricular K(ATP) channels enhances arrhythmogenicity, suggesting that such activation may contribute to reentrant arrhythmias in ischemic hearts.

Differential KATP channel pharmacology in intact mouse heart

Classically, cardiac sarcolemmal K(ATP) channels have been thought to be composed of Kir6.2 (KCNJ11) and SUR2A (ABCC9) subunits. However, the evidence is strong that SUR1 (sulfonylurea receptor type 1, ABCC8) subunits are also expressed in the heart and that they play a significant functional role in the atria. To examine this further, we have assessed the effects of isotype-specific potassium channel-opening drugs, diazoxide (specific to SUR1>SUR2A) and pinacidil (SUR2A>SUR1), in intact hearts from wild-type mice (WT, n=6), SUR1(-/-) (n=6), and Kir6.2(-/-) mice (n=5). Action potential durations (APDs) in both atria and ventricles were estimated by optical mapping of the posterior surface of Langendorff-perfused hearts. To confirm the atrial effect of both openers, isolated atrial preparations were mapped in both WT (n=4) and SUR1(-/-) (n=3) mice. The glass microelectrode technique was also used to validate optical action potentials. In WT hearts, diazoxide (300 microM) decreased APD in atria (from 33.8+/-1.9 ms to 24.2+/-1.1 ms, p<0.001) but was without effect in ventricles (APD 60.0+/-7.6 ms vs. 60.8+/-7.5 ms, respectively, NS), consistent with an atrial-specific role for SUR1. The absence of SUR1 resulted in loss of efficacy of diazoxide in SUR1(-/-) atria (APD 36.8+/-1.9 ms vs. 36.8+/-2.8 ms, respectively, NS). In contrast, pinacidil (300 microM) significantly decreased ventricular APD in both WT and SUR1(-/-) hearts (from 60.0+/-7.6 ms to 29.8+/-3.5 ms in WT, p<0.001, and from 63.5+/-2.1 ms to 24.8+/-3.8 ms in SUR1(-/-), p<0.001), but did not decrease atrial APD in either WT or SUR1(-/-) hearts. Glibenclamide (10 microM) reversed the effect of pinacidil in ventricles and restored APD to control values. The absence of Kir6.2 subunits in Kir6.2(-/-) hearts resulted in loss of efficacy of both openers (APD 47.2+/-2.2 ms vs. 47.6+/-2.1 ms and 50.8+/-2.4 ms, and 90.6+/-5.7 ms vs. 93.2+/-6.5 ms and 117.3+/-6.4 ms, for atria and ventricle in control versus diazoxide and pinacidil, respectively). Collectively, these results indicate that in the same mouse heart, significant differential K(ATP) pharmacology in atria and ventricles, resulting from SUR1 predominance in forming the atrial channel, leads to differential effects of potassium channel openers on APD in the two chambers.

Localization of Na+ Channel Isoforms at the Atrioventricular Junction and Atrioventricular Node in the Rat

Background— The electrical activity of the atrioventricular node (AVN) is functionally heterogeneous, but how this relates to distinct cell types and the 3-dimensional structure of the AVN is unknown. To address this, we have studied the expression of Nav1.5 and other Na+ channel isoforms in the AVN. Methods and Results— The rat AVN was identified by Masson’s trichrome staining together with immunolabeling of marker proteins: connexin40, connexin43, desmoplakin, atrial natriuretic peptide, and hyperpolarization-activated and cyclic nucleotide–gated channel 4. Na+ channel expression was investigated with immunohistochemistry with isoform-specific Na+ channel antibodies. Nav1.1 was distributed in a similar manner to Nav1.5. Nav1.2 was not detected. Nav1.3 labeling was present in nerve fibers and cell bodies (but not myocytes) and was abundant in the penetrating atrioventricular (AV) bundle and the common bundle but was much less abundant in other regions. Nav1.5 labeling was abundant in the atrial and ventricular myocardium and the left bundle branch. Nav1.5 labeling was absent in the open node, penetrating AV bundle, AV ring bundle, and common bundle but present at a reduced level in the inferior nodal extension and transitional zone. Nav1.6 was not detected. Conclusions— Our findings provide molecular evidence of multiple electrophysiological cell types at the AV junction. Impaired AV conduction as a result of mutations in or loss of Nav1.5 must be the result of impaired conduction in the AVN inputs (inferior nodal extension and transitional zone) or output (bundle branches) rather than the AVN itself (open node and penetrating AV bundle).

Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex

AIMS Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. METHODS AND RESULTS In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca(2+) cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2(-/-)) mice. Casq2(-/-) mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca(2+) cycling, including abnormal Ca(2+) release, periods of significantly elevated diastolic Ca(2+) levels leading to pauses and unstable pacemaker rate. Importantly, Ca(2+) cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca(2+) transient upstrokes throughout the atrial pacemaker complex. CONCLUSIONS Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca(2+) release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients.

Conduction barriers and pathways of the sinoatrial pacemaker complex: their role in normal rhythm and atrial arrhythmias

Since Keith and Flacks anatomical discovery of the sinoatrial node (SAN), the primary pacemaker of the heart, the question of how such a small SAN structure can pace the entire heart has remained for a large part unanswered. Recent advances in optical mapping technology have made it possible to unambiguously resolve the origin of excitation and conduction within the animal and human SAN. The combination of high-resolution optical mapping and histological structural analysis reveals that the canine and human SANs are functionally insulated from the surrounding atrial myocardium, except for several critical conduction pathways. Indeed, the SAN as a leading pacemaker requires anatomical (fibrosis, fat, and blood vessels) and/or functional barriers (paucity of connexins) to protect it from the hyperpolarizing influence of the surrounding atrium. The presence of conduction barriers and pathways may help explain how a small cluster of pacemaker cells in the SAN pacemaker complex manages to depolarize different, widely distributed areas of the right atria as evidenced functionally by exit points and breakthroughs. The autonomic nervous system and humoral factors can further regulate conduction through these pathways, affecting pacemaker automaticity and ultimately heart rate. Moreover, the conduction barriers and multiple pathways can form substrates for reentrant activity and thus lead to atrial flutter and fibrillation. This review aims to provide new insight into the function of the SAN pacemaker complex and the interaction between the atrial pacemakers and the surrounding atrial myocardium not only in animal models but also human hearts.