A peptide mimetic of the connexin43 carboxyl terminus reduces gap junction remodeling and induced arrhythmia following ventricular injury.

RATIONALE: Remodeling of connexin (Cx)43 gap junctions (GJs) is linked to ventricular arrhythmia. OBJECTIVES: A peptide mimetic of the carboxyl terminal (CT) of Cx43, incorporating a postsynaptic density-95/disks-large/ZO-1 (PDZ)-binding domain, reduces Cx43/ZO-1 interaction and GJ size remodeling in vitro. Here, we determined: (1) whether the Cx43-CT mimetic αCT1 altered GJ remodeling following left ventricular (LV) injury in vivo; (2) whether αCT1 affected arrhythmic propensity; and (3) the mechanism of αCT1 effects on arrhythmogenicity and GJ remodeling. METHODS AND RESULTS: A cryoinjury model generating a reproducible wound and injury border zone (IBZ) in the LV was used. Adherent methylcellulose patches formulated to locally release αCT1 (< 48 hours) were placed on cryoinjuries. Relative to controls, Cx43/ZO-1 colocalization in the IBZ was reduced by αCT1 by 24 hours after injury. Programmed electric stimulation ex vivo and optical mapping of voltage transients indicated that peptide-treated hearts showed reduced inducible arrhythmias and increased ventricular depolarization rates 7 to 9 days after injury. At 24 hours and 1 week after injury, αCT1-treated hearts maintained Cx43 in intercalated disks (IDs) in the IBZ, whereas by 1 week after injury, controls demonstrated Cx43 remodeling from IDs to lateralized distributions. Over a postinjury time course of 1 week, αCT1-treated IBZs showed increased Cx43 phosphorylation at serine368 (Cx43-pS368) relative to control tissues. In biochemical assays, αCT1 promoted phosphorylation of serine368 by protein kinase (PK)C-ε in a dose-dependent manner that was modulated by, but did not require ZO-1 PDZ2. CONCLUSIONS: αCT1 increases Cx43-pS368 in vitro in a PKC-ε-dependent manner and in the IBZ in vivo acutely following ventricular injury. αCT1-mediated increase in Cx43-pS368 phosphorylation may contribute to reductions in inducible-arrhythmia following injury.

Spatiotemporal induction of matrix metalloproteinase-9 transcription after discrete myocardial injury.

Radiofrequency (RF) ablation of the myocardium causes discrete sites of injury. RF scars can expand, altering the extracellular matrix (ECM) structure and the continuity of the electrical syncytium of the adjacent myocardium. Matrix metalloproteinases (MMPs), such as MMP-9, contribute to ECM remodeling. However, whether and to what degree transcriptional induction of MMP-9 occurs after myocardial RF injury and the association with electrical conduction patterns after RF injury remains unexplored. This study examined MMP-9 gene promoter (M9PROM) activation after myocardial RF injury using mice in which the M9PROM was fused to a ß-galactosidase (ß-gal) reporter. RF lesions (0.5-mm probe, 80°C, 30 s) were created on the left ventricular (LV) epicardium of M9PROM mice (n=62) and terminally studied at 1 h, 1 d, 3 d, 7 d, 14 d, and 28 d after RF injury. M9PROM activation was localized through ß-gal staining. The RF scar area and the area of ß-gal staining were measured and normalized to LV area (planimetry). RF scar size increased from 1 h post-RF-injury values by 7 d and remained higher at 28 d. M9PROM activation became evident at 3 d and peaked at 7 d. Electrical conduction was measured (potentiometric dye mapping) at 7 d after RF injury. Heterogeneities in action potentials and electrical impulse propagation coincident with M9PROM activation were observed after RF injury. For example, conduction proximal to the RF site was slower than that in the remote myocardium (0.15±0.02 vs. 0.83±0.08 mm/ms, P<0.05). Thus, a unique spatiotemporal pattern of MMP-9 transcriptional activation occurred after discrete myocardial injury, which was associated with the development of electrical heterogeneity. Therefore, these findings suggest that changes in a key determinant of extracellular matrix remodeling, in addition to changes in myocardial structure, can contribute to arrhythmogenesis around the region of myocardial injury.

Matrix metalloproteinase-7 affects connexin-43 levels, electrical conduction, and survival after myocardial infarction.

BACKGROUND: Matrix metalloproteinases (MMPs) contribute to left ventricular remodeling after myocardial infarction (MI). Specific causative roles of particular MMPs, however, remain unclear. MMP-7 is abundant in cardiomyocytes and macrophages, but MMP-7 function after MI has not been defined. METHODS AND RESULTS: Wild-type (WT; n=55) and MMP-7-null (MMP-7-/-; n=32) mice underwent permanent coronary artery ligation for 7 days. MI sizes were similar, but survival was greatly improved in MMP-7-/- mice. The survival difference could not be attributed to differences in left ventricular dilation because end-diastolic volumes increased similarly. ECG analysis revealed a prolonged PR interval in WT but not in MMP-7-/- post-MI mice. Post-MI conduction velocity, determined by optically mapping electrical wavefront propagation, decreased to 78+/-6% of control for WT and was normalized in MMP-7-/- mice. In WT mice, slower conduction velocity correlated with a 53% reduction in the gap junction protein connexin-43. Direct binding of MMP-7 to connexin-43, determined by surface plasmon resonance technology, occurred in a dose-dependent manner. Connexin-43 processing by MMP-7 was confirmed by in silico and in vitro substrate analyses and MMP-7 infusion induced arrhythmias in vivo. CONCLUSIONS: MMP-7 deletion results in improved survival and myocardial conduction patterns after MI. This is the first report to implicate MMP-7 in post-MI remodeling and to demonstrate that connexin-43 is a novel MMP-7 substrate.

Knockout of the neural and heart expressed gene HF-1b results in apical deficits of ventricular structure and activation.

OBJECTIVE: Knockout of the neural and cardiac expressed transcription factor HF-1b causes electrophysiological abnormalities including fatal ventricular arrhythmias that occur with increasing frequency around the 4th week of postnatal life. This study addresses factors that may contribute to conduction disturbance in the ventricle of the HF-1b knockout mouse. Disruptions to gap junctional connexin40 (Cx40) have been reported in distal (i.e., apically located), but not proximal His-Purkinje conduction tissues of the HF-1b knockout mouse. This abnormality in myocardial Cx40 led us to address whether 4-week-old HF-1b knockout postnates display other disruptions to ventricular structure and function. METHODS: Western blotting and immunoconfocal quantification of Cx43 and coronary arteriole density and function were undertaken in the ventricle. Electrical activation was described by optical mapping. RESULTS: Western blotting and immunoconfocal microscopy indicated that overall levels of Cx43 (p<0.001) and percent of Cx43 localized in intercalated disks (p<0.001) were significantly decreased in the ventricular myocardium of knockouts relative to wildtype littermate controls. Analysis of the reduction in Cx43 level by basal and apical territories revealed that the decrease was most pronounced in the lower, apical half of the ventricle of knockouts relative to controls (p<0.001). Myocyte size also showed a significant decrease in the knockout, that was more marked within the apical half of the ventricle (p<0.05). Optical recordings of ventricular activation indicated apically localized sectors of slowed conduction in knockout ventricles not occurring in controls that could be correlated directly to tissues showing reduced Cx43. These discrete sectors of abnormal conduction in the knockout heart were resolved following point stimulation of the ventricular epicardium and thus were not explained by dysfunction of the His-Purkinje system. To further probe base-to-apex abnormalities in the HF-1b knockout ventricle, we analyzed coronary arterial structure and function. These analyses indicated that relative to controls, the apical ventricular territory of the HF-1b knockout had reductions in the density of small resistance vessels (p<0.01) and deficits in arterial function as assayed by bead perfusion (p<0.01). CONCLUSION: The HF-1b knockout ventricle displays abnormalities in Cx43 level, myocyte size, activation spread and coronary arterial structure and function. These abnormalities tend to be more pronounced in the apical territory of the ventricle and seem likely to be factors contributing to the pathological disturbance of cardiac conduction that characterizes the heart of the HF-1b knockout mouse.

Cardiac neural crest ablation inhibits compaction and electrical function of conduction system bundles.

Retroviral and transgenic lineage-tracing studies have shown that neural crest cells associate with the developing bundles of the ventricular conduction system. Whereas this migration of cells does not provide progenitors for the myocardial cells of the conduction system, the question of whether neural crest affects the differentiation and/or function of cardiac specialized tissues continues to be of interest. Using optical mapping of voltage-sensitive dye, we determined that ventricles from chick embryos in which the cardiac neural crest had been laser ablated did not progress to apex-to-base activation by the expected stage [i.e., Hamburger and Hamilton (HH) 35] but instead maintained basal breakthroughs of epicardial activation consistent with immature function of the conduction system. In direct studies of activation, waves of depolarization originating from the His bundle were found to be uncommon in control hearts from HH34 and HH35 embryos. However, activations propagating from septal base, at or near the His bundle, occurred frequently in hearts from HH34 and HH35 neural crest-ablated embryos. Consistent with His bundle cells maintaining electrical connections with adjacent working myocytes, histological analyses of hearts from neural crest-ablated embryos revealed His bundles that had not differentiated a lamellar organization or undergone a process of compaction and separation from surrounding myocardium observed in controls. Furthermore, measurements on histological sections from optically mapped hearts indicated that, whereas His bundle diameter in control embryos thinned by almost one-half between HH30 and HH34, the His bundle in ablated embryos underwent no such compaction in diameter, maintaining a thickness at HH30, HH32, and HH34 similar to that observed in HH30 controls. We conclude that the cardiac neural crest is required in a novel function involving lamellar compaction and electrical isolation of the basally located His bundle from surrounding myocardium.

Nkx2.5 cell-autonomous gene function is required for the postnatal formation of the peripheral ventricular conduction system.

The ventricular conduction system is responsible for rapid propagation of electrical activity to coordinate ventricular contraction. To investigate the role of the transcription factor Nkx2.5 in the morphogenesis of the ventricular conduction system, we crossed Nkx2.5(+/-) mice with Cx40(eGFP/+) mice in which eGFP expression permits visualization of the His-Purkinje conduction system. Major anatomical and functional disturbances were detected in the His-Purkinje system of adult Nkx2.5(+/-)/Cx40(eGFP/+) mice, including hypoplasia of eGFP-positive Purkinje fibers and the disorganization of the Purkinje fiber network in the ventricular apex. Although the action potential properties of the individual eGFP-positive cells were normal, the deficiency of Purkinje fibers in Nkx2.5 haploinsufficient mice was associated with abnormalities of ventricular electrical activation, including slowed and decremented conduction along the left bundle branch. During embryonic development, eGFP expression in the ventricular trabeculae of Nkx2.5(+/-) hearts was qualitatively normal, with a measurable deficiency in eGFP-positive cells being observed only after birth. Chimeric analyses showed that maximal Nkx2.5 levels are required cell-autonomously. Reduced Nkx2.5 levels are associated with a delay in cell cycle withdrawal in surrounding GFP-negative myocytes. Our results suggest that the formation of the peripheral conduction system is time- and dose-dependent on the transcription factor Nkx2.5 that is cell-autonomously required for the postnatal differentiation of Purkinje fibers.

Changes in activation sequence of embryonic chick atria correlate with developing myocardial architecture.

To characterize developmental changes in impulse propagation within atrial musculature, we performed high-speed optical mapping of activation sequence of the developing chick atria using voltage-sensitive dye. The activation maps were correlated with detailed morphological studies using scanning electron microscopy, histology, and whole mount confocal imaging with three-dimensional reconstruction. A preferential pathway appeared during development within the roof of the atria, transmitting the impulse rapidly from the right-sided sinoatrial node to the left atrium. The morphological substrate of this pathway, the bundle of Bachman, apparent from stage 29 onward, was a prominent ridge of pectinate muscles continuous with the terminal crest. Further acceleration of impulse propagation was noted along the ridges formed by the developing pectinate muscles, ramifying from the terminal crest toward the atrioventricular groove. In contrast, when the impulse reached the interatrial septum, slowing was often observed, suggesting that the septum acts as a barrier or sink for electrical current. We conclude that these inhomogeneities in atrial impulse propagation are consistent with existence of a specialized network of fast-conducting tissues. The purpose of these preferential pathways appears to be to assure synchronous atrial activation and contraction rather than rapid impulse conduction between the sinoatrial and atrioventricular nodes.

Development of the cardiac pacemaking and conduction system.

The heartbeat is initiated and coordinated by a heterogeneous set of tissues, collectively referred to as the pacemaking and conduction system (PCS). While the structural and physiological properties of these specialized tissues has been studied for more than a century, distinct new insights have emerged in recent years. The tools of molecular biology and the lessons of modern embryology are beginning to uncover the mechanisms governing induction, patterning and developmental integration of the PCS. In particular, significant advances have been made in understanding the developmental biology of the fast conduction network in the ventricles--the His-Purkinje system. Although this progress has largely been made by using animal models such as the chick and mouse, the insights gained may help explain cardiac disease in humans, as well as lead to new treatment strategies.

Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart.

Impulse-conducting Purkinje fibers differentiate from myocytes during embryogenesis. The conversion of contractile myocytes into conduction cells is induced by the stretch/pressure-induced factor, endothelin (ET). Active ET is produced via proteolytic processing from its precursor by ET-converting enzyme 1 (ECE1) and triggers signaling by binding to its receptors. In the embryonic chick heart, ET receptors are expressed by all myocytes, but ECE1 is predominantly expressed in endothelial cells of coronary arteries and endocardium along which Purkinje fiber recruitment from myocytes takes place. Furthermore, co-expression of exogenous ECE1 and ET-precursor in the embryonic heart is sufficient to ectopically convert cardiomyocytes into Purkinje fibers. Thus, localized expression of ECE1 defines the site of Purkinje fiber recruitment in embryonic myocardium. However, it is not known how ECE1 expression is regulated in the embryonic heart. The unique expression pattern of ECE1 in the embryonic heart suggests that blood flow-induced stress/stretch may play a role in patterning ECE1 expression and subsequent induction of Purkinje fiber differentiation. We show that gadolinium, an antagonist for stretch-activated cation channels, downregulates the expression of ECE1 and a conduction cell marker, Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway. Conversely, pressure-overload in the ventricle by conotruncal banding results in a significant expansion of endocardial ECE1 expression and Cx40-positive putative Purkinje fibers. Coincident with this, an excitation pattern typical of the mature heart is precociously established. These in vivo data suggest that biomechanical forces acting on, and created by, the cardiovascular system during embryogenesis play a crucial role in Purkinje fiber induction and patterning.