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.

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.

Calcium gradients in conifer pollen tubes; dynamic properties differ from those seen in angiosperms.

Pollen tubes are an established model system for examining polarized cell growth. The focus here is on pollen tubes of the conifer Norway spruce (Picea abies, Pinaceae); examining the relationship between cytosolic free Ca2+, tip elongation, and intracellular motility. Conifer pollen tubes show important differences from their angiosperm counterparts; they grow more slowly and their organelles move in an unusual fountain pattern, as opposed to reverse fountain, in the tip. Ratiometric ion imaging of growing pollen tubes, microinjected with fura-2-dextran, reveals a tip-focused [Ca2+]i gradient extending from 450 nM at the extreme apex to 225 nM at the base of the tip clear zone. Injection of 5,5' dibromo-BAPTA does not dissipate the apical gradient, but stops cell elongation and uniquely causes rapid, transient increases of apical free Ca2+. The [Ca2+]i gradient is, however, dissipated by reversible perfusion of extracellular caffeine. When the basal cytosolic free Ca2+ concentration falls below 150 nM, again a large increase in apical [Ca2+]i occurs. An external source of calcium is not required for germination but significantly enhances elongation. However, both germination and elongation are significantly inhibited by the inclusion of calcium channels blockers, including lanthanum, gadolinium, or verapamil. Modulation of intracellular calcium also affects organelle position and motility. Extracellular perfusion of lanthanides reversibly depletes the apical [Ca2+]i gradient, altering organelle positioning in the tip. Later, during recovery from lanthanide perfusion, organelle motility switches direction to a reverse fountain. When taken together these data show a unique interplay in Picea abies pollen tubes between intracellular calcium and the motile processes controlling cellular organization.

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.

Microtubules and microfilaments coordinate to direct a fountain streaming pattern in elongating conifer pollen tube tips.

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-microm-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.

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.