Kenneth W. Hewett

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.

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 embyogenesis play a crucial role in Purkinje fiber induction and patterning.

Alterations in myocyte shape and basement membrane attachment with tachycardia-induced heart failure.

Chronic supraventricular tachycardia (SVT) results in left ventricular (LV) dilatation and dysfunction. However, the underlying mechanisms responsible for LV failure in this setting are not known. LV force production is dependent on the coupling of myocytes to the extracellular matrix, which is mediated through the basement membrane. This study was designed to determine whether alterations in myocyte geometry and basement membrane attachment are associated with LV failure in a pacing-induced model of cardiomyopathy. Echocardiographic measurement of LV function was performed in six pigs after 3 weeks of pacing-induced SVT (240 beats/min) and in eight sham-operated controls. Myocytes from these hearts were isolated, and attachment studies to specific components of the basement membrane were performed using laminin, fibronectin, and collagen IV. The SVT group when compared with the control group showed a significant reduction of LV fractional shortening (14 +/- 2% versus 31 +/- 2%, respectively; p less than 0.05), increased end-diastolic dimension (50 +/- 1 versus 35 +/- 1 mm, respectively; p less than 0.05), and lengthening of isolated myocytes (196 +/- 18 versus 142 +/- 9 microns, respectively; p less than 0.05). Myocyte attachment to laminin (50 micrograms/ml) was significantly decreased at 60 minutes in the SVT group compared with the control group (18.2 +/- 4.5 versus 60.9 +/- 4.5 cells/mm2, respectively; p less than 0.05). Similar reductions in myocyte attachment to fibronectin and collagen IV were observed. Ultrastructural examination of LV sections revealed focal disruptions of the basement membrane-sarcolemmal interface and a reduced number of sarcolemmal festoons in SVT hearts compared with control hearts (0.8 +/- 0.6 versus 2.8 +/- 0.8/4 microns, respectively; p less than 0.05). These alterations in myocyte morphology and basement membrane attachment may contribute to the LV failure associated with chronic SVT. Further, these structural changes may play a significant role in the progression of ventricular dysfunction as well as recovery from chronic SVT.

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 &agr;CT1 altered GJ remodeling following left ventricular (LV) injury in vivo; (2) whether &agr;CT1 affected arrhythmic propensity; and (3) the mechanism of &agr;CT1 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 &agr;CT1 (<48 hours) were placed on cryoinjuries. Relative to controls, Cx43/ZO-1 colocalization in the IBZ was reduced by &agr;CT1 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, &agr;CT1-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, &agr;CT1-treated IBZs showed increased Cx43 phosphorylation at serine368 (Cx43-pS368) relative to control tissues. In biochemical assays, &agr;CT1 promoted phosphorylation of serine368 by protein kinase (PK)C-&egr; in a dose-dependent manner that was modulated by, but did not require ZO-1 PDZ2. Conclusions: &agr;CT1 increases Cx43-pS368 in vitro in a PKC-&egr;–dependent manner and in the IBZ in vivo acutely following ventricular injury. &agr;CT1-mediated increase in Cx43-pS368 phosphorylation may contribute to reductions in inducible-arrhythmia following injury.

Changes in L-type calcium channel abundance and function during the transition to pacing-induced congestive heart failure

OBJECTIVE The development of congestive heart failure (CHF) is accompanied by left ventricular (LV) and myocyte contractile dysfunction. However, time-dependent cellular and ionic events which contribute to the initiation and progression of CHF remain unclear. This study tested the central hypothesis that changes in L-type Ca2+ channel current (ICa) and abundance (Bmax) are early events in the transition to CHF. METHODS LV fractional shortening by echocardiography, isolated LV myocyte shortening velocity by videomicroscopy, ICa by voltage-clamp, and Bmax by [3H]nitrendipine binding were determined at each week during the progression of pacing-induced CHF in pigs (240 bpm; n = 6/week for 3 weeks). Myocyte and L-type Ca2+ channel function were determined under basal conditions and after beta-adrenergic receptor stimulation with 25 nM isoproterenol. RESULTS After 1 week of pacing, myocyte and L-type Ca2+ current responses to beta-adrenergic receptor stimulation were reduced by 20% from control values and was accompanied by over a 210% increase in plasma catecholamine levels. After 2 weeks of pacing, reductions in LV fractional shortening and myocyte shortening velocity from control values (20 +/- 1 vs. 34 +/- 2% and 36.7 +/- 2.9 vs. 50.6 +/- 2.4 microns/s, respectively, P < 0.05) were paralleled by decreased ICa (2.47 +/- 0.10 vs. 3.63 +/- 0.25 pA/pF, P < 0.02) and Bmax (149 +/- 16 vs. 180 +/- 12 fmol/mg, P < 0.03). After 3 weeks of pacing, LV fractional shortening was reduced by over 50%, myocyte shortening velocity by 37%, and ICa and Bmax were reduced by over 25% from control values. Furthermore, after 3 weeks of pacing, the ICa/Bmax ratio was reduced from control values (16.2 +/- 0.9 vs. 20.6 +/- 1.2 [fA/pF]/[fmol/mg], P < 0.03), which suggests functional defects in the remaining L-type Ca2+ channels. CONCLUSIONS An early event during the transition to pacing-induced CHF was diminished beta-adrenergic receptor augmented L-type Ca2+ current, which was followed by an absolute loss of steady-state L-type Ca2+ current and channel abundance. The development of severe CHF was accompanied by a loss of Ca2+ carrying capacity through residual channels. These unique findings suggest that a contributory molecular mechanism for the initiation and progression of CHF is changes in the structure and function of the L-type Ca2+ channels.

Myocyte electrophysiological properties following the development of supraventricular tachycardia-induced cardiomyopathy.

Chronic supraventricular tachycardia (SVT) causes left ventricular (LV) dilatation and dysfunction, diminished myocyte contractile function, and abnormalities in sarcolemmal receptor systems. We hypothesized that changes in myocyte action potential characteristics and L-type Ca2+ channel (Ca2+ channel) function, which are major determinants of myocyte contractile processes, would occur with SVT cardiomyopathy. LV function and isolated myocyte contractile function were examined in 11 pigs with SVT cardiomyopathy (pace 240 bpm; 3 weeks) and 11 control pigs. With chronic SVT, LV fractional shortening fell and myocyte shortening velocity was reduced compared to controls (11 +/- 2 v 37 +/- 2%, P < 0.0001; and 32.5 +/- 1.2 v 55.7 +/- 1.6 microns/s, P < 0.0001, respectively). Isolated myocyte action potential upstroke velocity and amplitude were reduced with SVT cardiomyopathy compared to controls (92.8 +/- 4.8 v 129.5 +/- 3.1 V/s, P < 0.0001; and 98.2 +/- 2.2 v 110.3 +/- 1.3 mV, P < 0.0001, respectively). the duration of the myocyte action potential, defined as the time to 90% repolarization, was prolonged with SVT cardiomyopathy compared to controls (201.7 +/- 5.9 v 169.1 +/- 6.8 ms, P = 0.002). These specific abnormalities in the indices of myocyte contractile function and action potential characteristics which occurred with SVT cardiomyopathy were not normalized following beta-adrenergic receptor stimulation. In order to determine a potential mechanism for the changes in myocyte contractile function and action potential characteristics with SVT cardiomyopathy, Ca2+ channel function was examined in control and SVT myocytes. In SVT myocytes, peak L-type Ca2+ current (ICa) normalized to membrane capacitance and the Ca2+ channel inactivation time constant were reduced compared to controls (-2.30 +/- 0.24 v -3.79 +/- 0.28 pA/pF, P = 0.0001; and 104.0 +/- 10.8 v 199.9 +/- 27.4 ms, P = 0.005, respectively). The abnormalities in Ca2+ channel function with SVT cardiomyopathy persisted in myocytes with equivalent membrane capacitances and were not normalized with beta-adrenergic receptor stimulation. In conclusion, findings from the present study suggest that fundamental abnormalities in myocyte electrical events (action potential) and ionic flux (Ca2+ channel function) are contributory mechanisms for the depressed myocyte contractile function with SVT cardiomyopathy.

The rate and anisotropy of impulse propagation in the postnatal terminal crest are correlated with remodeling of Cx43 gap junction pattern

BACKGROUND Disruptions to intermyocyte coupling have been implicated in arrhythmogenesis and development of conduction disturbances. At present, understanding of the relationship between the microscopic organization of intercellular coupling and the macroscopic spread of impulse in the normal and diseased heart is largely confined to theoretical analyses. METHODS AND RESULTS The abundance and arrangement of gap junctions, as well as conduction properties, were assessed in terminal crest preparations isolated from the atria of neonate, weanling, and adult rabbits. We report that the connexin composition of terminal crest was uncomplicated, with Cx43 being the most prominent isoform detectable by Western blotting and immunostaining. Terminal crest myocytes showed little change in total Cx43-gap junction per cell during postnatal growth as assessed by stereology. However, marked non-uniformities emerged in the sarcolemmal distribution of Cx43-gap junctions. Cx43-gap junction area at myocyte termini increased 3.5-fold from birth to adulthood. Correlated with this change in Cx43, impulse propagation velocity parallel to the myofiber axis, as assessed by multi-site optical mapping using voltage-sensitive dye (di-4-ANEPPS), increased 2.4-fold. Conversely, the amount of Cx43-gap junctions on myocyte sides, and the conduction velocity transverse to the myofiber axis, remained relatively invariant during maturation. Hence, the increasing electrical anisotropy of maturing terminal crest was wholly accounted for by increases in conductance velocity along the bundle. This increase in longitudinal conduction velocity was correlated with changes in the sarcolemmal pattern, but not the overall density, of Cx43-gap junctions. CONCLUSIONS This study provides the first correlative structure/function analysis of the relationship between the macroscopic conduction of impulse and the microscopic cellular organization of gap junctions in a differentiating cardiac bundle. Confirmation is provided for theoretical predictions which emphasize the importance of the cell-to-cell geometry of coupling in determining the spread and pattern of myocardial activation.

The direct effects of protamine sulfate on myocyte contractile processes

The use of protamine sulfate in patients has been associated with circulatory collapse and is suspected to directly depress left ventricular function. However, the cellular basis for these changes that occur after protamine administration are unknown. Accordingly, the first objective of this study was to determine the direct effects of protamine on isolated myocyte contractile function. Myocytes were isolated from porcine hearts and contractile function was examined at baseline and then after the administration of protamine in concentrations of 20, 40, or 80 micrograms/ml. These concentrations were chosen because they reflect the serum concentrations of protamine commonly obtained in patients. The presence of protamine resulted in a dose-dependent decline in myocyte contractile function. For example, in the presence of a 20 microgram/ml concentration of protamine myocyte contractile function did not change significantly from baseline values, whereas an 80 microgram/ml protamine concentration caused myocyte percent and velocity of shortening to fall by more than 35% from baseline values. In light of the fact that protamine directly depressed myocyte contractile function, a second objective of this study was to examine potential cellular mechanisms responsible for this effect. Accordingly, in the next series of experiments, the effects of protamine on the myocyte sarcolemmal beta-adrenergic receptor system were examined by measuring myocyte contractile function with the beta-adrenergic agonist isoproterenol (25 nmol/L), as well as with the concomitant addition of protamine and isoproterenol. In the presence of protamine, myocyte beta-adrenergic responsiveness was significantly reduced. For example, in the presence of an 80 microgram/ml dose of protamine, both myocyte percent and velocity of shortening fell by greater than 50% when compared with isoproterenol alone values (p < 0.05). To determine the reversibility of these protamine effects, we performed additional experiments in the presence of heparin. Incubation with heparin before protamine addition prevented the negative effects of protamine on myocyte function. However, the addition of heparin after protamine incubation failed to reverse the negative effects of protamine on myocyte function. In a final set of experiments, the effects of protamine on isolated myocyte electrophysiologic properties were examined using microelectrode techniques at baseline and with either 40 or 80 micrograms/ml doses of protamine. Myocyte resting membrane potential changed from baseline with the addition of a 40 micrograms/ml dose of protamine (-79.2 +/- 0.5 versus -75.2 +/- 0.8 mV (p < 0.05), with no further change at an 80 micrograms/ml dose of protamine (-73.0 +/- 1.3 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

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 β‐galactosidase (β‐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 β‐gal staining. The RF scar area and the area of β‐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.—Mukherjee, R., Colbath, G. P., Justus, C. D., Bruce, J. A., Allen, C. M., Hewett, K. W., Saul, J. P., Gourdie, R. G., Spinale, F. G. Spatiotemporal induction of matrix metalloproteinase‐9 transcription following discrete myocardial injury. FASEB J. 24, 3819–3828 (2010). www.fasebj.org

Inducible lethal ventricular arrhythmias in swine with pacing-induced heart failure

AbstractIntroduction: Rapid pacing-induced heart failure provides an excellent animal model for the study of heart failure. We studied the development of ventricular tachyarrhythmias using programmed stimulation in a pacing-induced heart failure model. We also studied action potential characteristics and the relationship between action potential and heart rate. Methods and results: Ten pigs were instrumented and were studied before the onset and every week after rapid pacing was instituted. Weekly echocardiograms and programmed stimulation were done in a sedated state. In vitro electrophysiologic studies were done on left ventricular myocardium in 4 heart-failure animals and 4 controls. All animals developed progressive heart failure with left ventricular dilatation and reduced percentage fractional shortening. No arrhythmias were induced at baseline or the first and second weeks. Ventricular fibrillation was induced in one animal on the third week and 4 animals on week 4, while there was no appreciable worsening in echocardiographic indices of ventricular dysfunction between weeks 3 and 4. Ventricular effective refractory period was unchanged during the 4 weeks. In vitro studies showed action potential prolongation in heart failure myocardium. However, action potential duration at pacing rates > 100 bpm were similar to controls. No early or delayed afterdepolarizations were observed. Conclusion: This study demonstrated an increased susceptibility to ventricular fibrillation with the development of heart failure which was not related to the degree of ventricular disfunction. Also, the normalization of action potential duration at higher heart rates suggests that the increased incidence of inducible ventricular fibrillation in this model may not be solely due to prolonged action potential duration.