• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Circulation. Author manuscript; available in PMC Jul 6, 2011.
Published in final edited form as:
PMCID: PMC2946370
NIHMSID: NIHMS213444

Chronic Localized High Frequency Electrical Stimulation Within the Myocardial Infarct: Effects on Matrix Metalloproteinases and Regional Remodeling

Abstract

Background

Disruption of the balance between matrix metalloproteinases (MMP) and MMP inhibitors (TIMPs) within a myocardial infarct (MI) contribute to left ventricular (LV) wall thinning and changes in regional stiffness at the MI region. This study tested the hypothesis that a targeted regional approach through localized high frequency stimulation (LHFS) using low amplitude electrical pulses instituted within a formed MI scar would alter MMP/TIMP levels and prevent MI thinning.

Methods/Results

At 3 wks following MI, pigs were randomized for LHFS (n=7, 240bpm, 0.8V, 0.05ms pulses) or unstimulated (UNSTIM, n=10). At 4 wks post-MI, LV wall thickness (echo, 0.89±0.07 vs 0.67±0.08 cm, p<0.05) and regional stiffness (piezoelectric crystals, 14.70±2.08 vs 9.11±1.24, p<0.05) were higher with LHFS than UNSTIM. In vivo interstitial MMP activity (fluorescent substrate cleavage, 943±59 vs 1210±72 units, p<0.05) in the MI region was lower with LHFS than in UNSTIM. In the MI region, MMP-2 levels were lower, while TIMP-1 and collagen levels were higher with LHFS than in UNSTIM (all p<0.05). Transforming growth factor-β (TGF-β) receptor 1 and phosphorylated SMAD-2/3 levels within the MI region were higher with LHFS than in UNSTIM. Electrical stimulation (4Hz) of isolated fibroblasts resulted in a reduction of MMP-2 and MT1-MMP levels, but increased TIMP-1 levels compared to unstimulated fibroblasts.

Conclusions

These unique findings demonstrate that LHFS of the MI region altered LV wall thickness and material properties, likely due to reduced regional MMP activity. Thus, LHFS may provide a novel means to favorably modify LV remodeling post-MI.

Keywords: Myocardial infarction, extracellular matrix, remodeling, matrix metalloproteinases

INTRODUCTION

Events following a myocardial infarction (MI) include left ventricular (LV) remodeling in terms of progressive chamber dilation and heterogeneous changes in the cellular and extracellular constituents of the LV myocardium.1-3 In addition, the formation of the fibrotic MI scar alters myocardial material properties, such as regional myocardial stiffness.4-6 Moreover, these changes in myocardial structure at the MI region are speculated to result in a progressive thinning of the MI region, which is termed as infarct expansion.4 Increased matrix metalloproteinase (MMP) abundance and discordant alterations in the balance between MMPs and endogenous tissue inhibitors of the metalloproteinases (TIMPs) have been implicated in structural sequelae to MI.2, 4, 5, 7 While systemic pharmacological approaches can attenuate global LV dilation post-MI, a targeted modulation that results in reversing the adverse remodeling of a “mature” MI scar in terms of normalizing the imbalance between MMP/TIMP levels as well as the material characteristics remains problematic.

In vivo and in vitro studies have demonstrated that electrical stimulation can modulate a number of factors relevant to tissue remodeling.8-15 For example, electrical stimulation of dermal wounds accelerates the wound healing response in terms of collagen deposition and wound contraction [review11]. Electrically stimulating cartilage explanted from patients with osteoarthritis resulted in increased collagen deposition and reduced mRNA expression of certain MMP types.12 In fibroblast cultures, cellular viability, migration, and rate of protein synthesis, including that of matrix proteins, have been shown to be increased with electrical stimulation.11 However, whether and to what degree in vivo electrical stimulation of the MI region, which contains a high population of fibroblasts, would alter remodeling within the MI region remained unknown. Accordingly, this study tested the hypothesis that localized high frequency stimulation (LHFS) instituted within a formed MI scar using low amplitude electrical pulses would reduce MMP activity and prevent progressive MI thinning.

METHODS

All animals were treated and cared for in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (National Research Council, Washington, 1996), and the protocol was approved by the Institutional Animal Care and Use Committee. Permanent coronary ligation was performed in mature pigs (Yorkshire, n=17, 25 kg, Hambone Farms, Orangeburg, SC) as previously described.4 Briefly, the LV was accessed through a left-sided thoracotomy and a pericardiectomy performed. MI was induced by direct ligation of the first two obtuse marginal arteries (OM1, OM2) at the origin from the circumflex coronary artery (4.0 Proline).4, 5

Determination of localized high frequency stimulation (LHFS) parameters

The main concern in determining the LHFS stimulation parameters pertained to the safety of being able to confine electrical activation to within the MI region, but not cause escape of the triggered impulses that could result in ventricular tachycardia. Accordingly, the pacemaker parameters used in the present study were determined in preliminary studies performed in 3 post-MI pigs. At terminal study on day 28 post-MI, an acute pacing protocol was instituted using a pacemaker lead sutured to the center of the MI region on the epicardial surface and connected to an external pacemaker (Medtronic). The pacemaker was activated using pulses with amplitudes ranging from 0.5 V to 1.5 V (in increments of 0.1 V) and pulse durations of 0.01, 0.05, and 0.10 ms. The resultant propagation of the introduced impulses was mapped by measuring pulse amplitude (PONEMAH, sensitivity of 5 mV) on the epicardial surface using a template in the polar coordinate system (radial intervals in increments of 0.5 cm up to 2.5 cm and angle increments of 45°). Pulses with an amplitude of 0.8 V and duration of 0.05 ms represented the largest combination of amplitude/duration parameters that was completely confined to the MI region (at distances of 2.0 cm from the stimulating electrode, Figure 1A) in all 3 pigs. Therefore, these pulse parameters were used for chronic LHFS for the MI pigs in the present study. The same pacing parameters resulted in the capture of the ventricular rhythm in two non-MI pigs. Accordingly, referent controls used in this study were non-MI pigs not subjected to chronic LHFS.

Figure 1
(A). Electrical mapping of pulse amplitude in preliminary studies showed that propagation of pulses with amplitudes of 0.8 V and duration of 0.05 ms was completely confined to the infarct region. (B). Representative lead II tracing showing the first derivative ...

Chronic LHFS model

MI was induced as described above and a shielded pacemaker lead was sutured onto the epicardium between OM1 and OM2, 2 cm below the circumflex artery (n=17). A pacemaker (EnPulse, Medtronic, Minneapolis, MN) was buried in a subcutaneous pocket and connected to the pacemaker lead, which was tunneled through a purse-string incision on the thoracic wall. Pacemaker capture of the LV was confirmed by transiently (<30 s) activating the pacemaker at a rate 20% higher than the intrinsic heart rate of the pig.

LV echocardiography measurements were performed (S4 probe, 3.5 MHz, spatial resolution: 800 μm, Agilent Sonos 5500) to determine LV wall thickness and volumes using the method-of-disks variant of Simpson's algorithm.16 Inter- and intra-observer coefficients of variance while using this system were 9% and 4%, respectively. In this study, all echocardiographic measurements were performed by a single observer (WTR), who was blinded to the group assignment of the pigs. Following echocardiography at 21 days post-MI, pacemakers in the pigs randomized to the LHFS group (n=7) were activated at 240 bpm using low amplitude and short duration pulses (VOO mode, 0.8V, 0.05 ms). ECG recordings were obtained to confirm that pacemaker activation did not cause ventricular tachycardia through capture of the ventricular rate (Figure 1B). Pacemakers in the other MI pigs (n=10) were left deactivated, and these pigs comprised the unstimulated (UNSTIM) group.

Myocardial function and microdialysis measurements

At 28 days post-MI, pacemaker stimulation of the LV in the LHFS group was reconfirmed in the ECG tracing, following which the pacemakers were deactivated. Repeat echocardiographic measurements were obtained in all pigs. The pigs were anesthetized and instrumented for hemodynamic measurements of arterial pressures, pulmonary artery pressures, and cardiac output. A sternotomy was performed and a vascular ligature placed around the inferior vena cava in order to perform transient caval occlusion. A calibrated microtipped transducer (7.5 F, Millar Instruments Inc, Houston, TX) was placed in the LV through a small apical stab wound. Piezoelectric crystals (2 mm, Sonometrics, Ontario) positioned in the central portion of the MI region to record regional LV dimensions and wall thickness at a sampling frequency of 1000 Hz (Pentium-Sonolab, Sonometrics).

Steady state hemodynamics measurements included systemic and pulmonary artery pressures, cardiac output, and LV pressures. Following steady-state measurements, LV pre-load was altered by sequential occlusion and release of the inferior vena cava and isochronal measurements of LV pressure and dimensions recorded. From the digitized pressure-dimension data, regional myocardial stiffness of the MI region was computed.5

For interstitial MMP activity measurements, microdialysis probes with a molecular weight cutoff of 20 kDa and an outer diameter of 0.5 mm were placed in the remote and MI regions (2 probes per pig). A previously validated fluorogenic substrate specific for MMP-1, 2, 3, 7, and 9 (Calbiochem, La Jolla, CA)17 at a concentration of 60 μmol/L was infused at a rate of 5 μL/min and allowed to equilibrate for 30 minutes. Dialysate returning from both probes was collected into amber microcentrifuge tubes at 30 minute intervals. Fluorescence from dialysate samples (100 μL, FLUOstar Galaxy, BMG Lab Technologies, Durham, NC) were read at an excitation wavelength of 280 nm and an emission wavelength of 360 nm.

For the purposes of obtaining reference control values, 5 age and weight matched pigs were instrumented to measure LV myocardial function and interstitial MMP activity.

Myocardial histological and biochemical measurements

Following the final set of measurements, the heart was removed and the LV divided into MI and remote regions. These myocardial sections were flash frozen for biochemistry or placed in formalin for histological staining.

LV sections (5 μm thick) were stained with picrosirius red and the relative collagen percent area in the MI and remote regions were determined using computer assisted morphometric methods as described previously.18 Sections were imaged on an inverted microscope (Axioskop-2, Zeiss) and the images were digitized (AxioCam MRc, Zeiss). Collagen content was determined from the digitized images as a percentage of total tissue area in a minimum of 5 random high power fields from each myocardial region of each pig.

Immunostaining was used to determine whether TIMPs colocalized with cells that stained positive for α-sarcomeric actin (marker for myocytes) and α-smooth muscle actin (marker for myofibroblasts) in sections from the MI region of UNSTIM pigs (n=2) and following LHFS (n=3).18 Sections from non-MI pigs (n=2) were stained identically and used as referent controls. Briefly, these myocardial sections were subjected to antigen unmasking (0.1 mg/ml proteinase K) and blocked for 1 hr at room temperature (3% normal goat serum and 1% bovine serum albumin). Following an overnight incubation at 4°C with primary antibodies (TIMP-1: Chemicon AB770, 1:100; α-sarcomeric actin: Sigma A2172, 1:400; α-smooth muscle actin: Sigma A5228, 1:400; α-sarcomeric actin: Sigma A2172, 1:400), slides were washed and incubated at room temperature with fluorochrome conjugated secondary antibodies (FITC conjugated goat-anti-rabbit IgG (for α-smooth muscle actin staining), Cy3 conjugated donkey-anti-mouse IgM (for α-sarcomeric actin staining), and Cy5 conjugated goat-anti-mouse IgM (for TIMP-1 staining), Jackson ImmunoResearch). Negative controls included the use of the secondary antibody only with pre-immune serum. Fluorescent images of the sections were captured using a laser confocal microscope (Leica TCS SP2, Exton PA). A minimum of 3 high-powered fields (63x objective) were imaged from each myocardial section and immunopositive staining for each antibody was quantitated as a percentage of the total area of each image.

Relative MMP-2 and MMP-9 levels were determined by gelatin zymography and levels of MMP-1, MMP-8, MMP-13, MT1-MMP, TIMP-1, TIMP-2, and TIMP-4 were determined by immunoblotting.4 Abundance of α-smooth muscle actin, vimentin, prolyl-4-hydroxylase, hyaluronan binding protein, transforming growth factor-β, TGF-β receptors R1 and R2, SMAD-2/3, and phosphorylated SMAD-2/3 were determined by immunoblotting.19 Positive controls were included in each gel as appropriate. The zymograms and immunoblots were digitized, and levels of all analytes were quantitated (Gel Pro Analyzer, Media Cybernetics) by 2-dimensional integrated optical density (IOD). Myeloperoxidase activity was determined using ELISA (R&D Systems).

In vitro electrical stimulation of fibroblasts

Fibroblasts were isolated from the LV free wall of non-MI pigs using the outgrowth technique.20, 21 Briefly, the LV myocardial samples were minced, transferred to cell culture flasks (75 cm2, Falcon), and allowed to adhere. Sterile growth medium was added to the flasks, and the cells were incubated under standard cell culture conditions (37°C; 21% O2, 5% CO2) with culture media consisting of fibroblast growth medium (FGM, C23010, Promocell, Heidelberg, Germany), 20% fetal bovine serum, and Promocell Supplement Mixture (C39315). After a two week incubation period, myocardial fibroblasts were scraped and transferred to 0.2% gelatin-coated (Sigma-Aldrich, St. Louis, MO) tissue culture flasks and grown to confluency.

For the electrical stimulation studies, confluent cultures from passage 2 were used. Fibroblasts were plated onto a 4-well chamber (plastic base) at a density of 6×105 cells/well. Once the fibroblasts were 80% confluent, the medium was changed to a serum-free medium, in which the cells incubated for 24 hours. The culture medium was replaced with fresh serum-free medium and carbon (graphite) electrodes were placed at the ends of each chamber.22 The fibroblasts were electrically stimulated using 5 ms, 2 mA, 4 V/cm pulses of alternating polarity in each chamber. The cells were stimulated at either 4 Hz or left unstimulated (0 Hz). Following 24 hours of stimulation, the cell media was collected. The cells were trypsinized and cell count from each well (hemocytometer) was recorded. The cells were then centrifuged and the cell pellets resuspended in an extraction buffer.

Relative MMP-2 and MMP-9 levels were examined using gelatin zymography from the media samples.4 The relative abundance of MT1-MMP (AB38971) in the cell pellets and TIMP-1 (AB8116) in the media samples were determined by immunoblotting.4 The zymograms and immunoblots were quantitated as above.

Data analysis

Data was collected in a blinded fashion and remained coded until the end of the study. Normality of data distribution for each variable was checked with the Shapiro-Wilk test. Non-parametric tests, as detailed below, were used for variables in which a normal distribution could not be assumed. Echocardiographic measurements of LV geometry and function were compared between the two MI groups using a two-way analysis of variance (ANOVA) model with time (21 days and 28 days post-MI) and treatment group (UNSTIM and LHFS) being the factors for the model. Single point measurements were compared between the control and MI groups using a one-way ANOVA. Following the ANOVA, post hoc pair-wise comparisons were performed using t-tests corrected for number of comparisons by Tukey's method (module “prcompw”, STATA). Differences in LV end-diastolic volume between post-MI days 21 and 28 were computed as a percentage change and compared between the Winsorized (robust) means of the two MI groups using the Mann-Whitney test.23 For the interstitial MMP activity measurements, biochemical, and morphometric studies, comparisons to reference control values were performed using a one way ANOVA. Comparisons between the MI groups, in which the treatment effects were group and region, were performed using two way ANOVA. The relationships between collagen content, LV regional myocardial stiffness, and relative levels of MMP-1, MMP-8, and MMP-13 were examined using least squares linear regression analysis. Comparisons of areas of positive immunostaining were compared between groups using the Kruskal-Wallis test. For the fibroblast studies, The IOD values recorded from the zymographic and immunoblot assays were normalized to the number of cells in each well. The change in MMP and TIMP levels from unstimulated values was determined as the ratio of values recorded at each frequency and that of the average value for the unstimulated wells. This normalization procedure resulted in the MMP and TIMP levels in the unstimulated (0 Hz) group being assigned a value of 100%. Comparison of MMP and TIMP levels between unstimulated and 4 Hz values was compared using a one-way ANOVA and comparisons to the unstimulated group were performed using a two-tailed, one-sample mean comparison test against the value of 100%. All statistical analyses were performed using the STATA statistical software package (v8.0, Statacorp, College Station, TX). Results are presented as Mean ± standard error of the mean (SEM). Two-tailed p-values of less than 0.05 were considered to be statistically significant.

RESULTS

Serial measurements

All 17 pigs entered into the study survived the initial instrumentation and MI induction. LV echocardiographic measurements recorded for the control pigs and at post-MI days 21 and 28 for the two MI groups are presented in Table 1. LV posterior wall thickness was reduced and septal wall thickness was increased at 21 days post-MI for the MI group that was left unstimulated (UNSTIM) or for the MI group with low amplitude, high frequency stimulation (LHFS), with no difference between groups. At 28 days post-MI, LV posterior wall thickness was reduced further in the UNSTIM group, but remained similar to 21 day values in the LHFS group. The change in LV end-diastolic volume from 21 days post-MI to 28 day post-MI was lower with LHFS compared to the UNSTIM group (3.2±2.6% vs. 12.9±5.3%, respectively, p=0.03). LV ejection fraction in the post-MI period was similar in both groups.

Table 1
Left Ventricular (LV) Geometry and Function at 21 days and 28 days Following Myocardial Infarction (MI): Effects of MI Only (UNSTIM) or with concomitant Localized High Frequency Stimulation (LHFS)

Post-MI day 28 terminal studies

At 28 days post-MI protocol, measurements of hemodynamics and regional LV geometry and function were performed using sonomicrometry. Hemodynamics and steady-state sonomicrometry recordings for the control and both MI groups are summarized in Table 2. Mean arterial pressure in both MI groups were similar to control values. Cardiac index and steady-state regional segmental shortening (Table 2) was reduced from reference control values in both MI groups. Within the MI region, LV regional myocardial stiffness was increased in both post-MI groups, but was higher with LHFS compared to UNSTIM (Figure 2A). Interstitial MMP activity (Figure 2B) was higher than control values in the MI region of the UNSTIM group, but was similar to control values with LHFS.

Figure 2
(A). Regional myocardial stiffness at 28 days post-MI was determined using piezoelectric crystals implanted within the MI and remote regions and through beat-to-beat analysis of the end-diastolic pressure – dimension relationship while altering ...
Table 2
Hemodynamic Parameters at 28 days Following Myocardial Infarction (MI): Effects of MI Only (UNSTIM) or with concomitant Localized High Frequency Stimulation (LHFS)

LV myocardial collagen content

Morphometrically determined percent collagen (Figure 3) was increased within the MI regions in both MI groups compared to reference control levels. Compared to the UNSTIM group, however, relative collagen volume fraction within the MI region was increased with LHFS. There was a significant correlation between LV collagen content and regional myocardial stiffness in the MI region (Figure 3B).

Figure 3
TOP: Representative photomicrographs of picrosirius red birefringence from the MI region of a pig in which the pacemaker was not activated (UNSTIM, left) and from one that underwent LHFS (right). Scale bars: 200 μm. BOTTOM: (A). Compared to control ...

LV myocardial biochemistry

LV profiles for the MMPs and TIMPs are shown in Figure 4. Compared to the UNSTIM group, MMP-2 levels were lower and there was a trend for lower MMP-1 levels (p=0.08) within the MI region of the MI+LHFS group. TIMP-1 levels were higher within the MI region with LHFS compared to the UNSTIM group. There was a significant inverse relationship between MMP-1 levels and collagen content (Figure 5A, p<0.05).

Figure 4
Levels of MMP-2, MMP-9, MT1-MMP, MMP-1, MMP-8, and MMP-13, TIMP-1, TIMP-2, and TIMP-4 in the remote and MI regions of pigs that were either left unstimulated (UNSTIM) or underwent LHFS. Representative zymograms or immunoblots from the non-MI reference ...
Figure 5
Relationship between collagen content and levels of the collagenases (MMP-1, MMP-8, and MMP-13) within the MI region. There was a significant negative relationship between collagen content and MMP-1 levels (y=−2.36x + 3297.05, r:−0.58, ...

Cell type markers

Representative images (Figure 6) from immunohistological analysis showed that low amounts of TIMP-1, which was localized to interstitial space between myocytes (α-sarcomeric actin positive cells stained in blue), could be detected within the viable myocardium from the non-MI control animals. The MI region in the UNSTIM and LHFS groups was devoid of α-sarcomeric actin staining, indicating an absence of myocytes. Nevertheless, TIMP-1 staining was clearly present within the MI region of both groups, and was colocalized with α-smooth muscle actin positive cells. However, the number of α-smooth muscle actin positive cells and TIMP-1 colocalization appeared to be higher with LHFS than in UNSTIM. Immunoblotting of the myocardium from the remote and MI regions revealed that levels of α-smooth muscle actin, vimentin, prolyl-4-hydroxylase, and hyaluronan binding protein were differentially higher with LHFS than UNSTIM values.

Figure 6
Representative immunostained confocal photomicrographs of the lateral free wall of referent control pig and the MI region from pigs that were either left unstimulated (UNSTIM) or with LHFS. The sections were stained for α-smooth muscle actin (red), ...

Transforming growth factor (TGF)-β pathway

Levels of TGF-β and TGF-β Receptor 1 (TGF-β R1) within the MI region were increased over control values and that of the remote regions for both the UNSTIM and LHFS groups (Figure 7). However, TGF-β R1 levels were differentially higher with LHFS compared to UNSTIM. Levels of SMAD-2/3 and phosphorylated SMAD-2/3 were higher than control levels or of the remote myocardium within the MI region of both groups. However, phosphorylated SMAD-2/3 levels were higher with LHFS compared to UNSTIM. Consequently, the ratio of phosphorylated SMAD-2/3 to total SMAD-2/3 within the MI region was higher with LHFS than in the UNSTIM group.

Figure 7
Levels of transforming growth factor (TGF)-β, TGF-β receptors R1 and R2, SMAD-2/3, and phosphorylated SMAD-2/3 (pSMAD-2/3) in the remote and MI regions of pigs that were either left unstimulated (UNSTIM) or underwent LHFS. Representative ...

Electrical stimulation of isolated fibroblasts

Two independent cultures of myocardial fibroblasts from each pig were either stimulated at 4 Hz or left unstimulated, for a total of n=6 experiments for the UNSTIM and 4 Hz groups. Following 24 hours of stimulation, the cells retained spindle-shaped morphology (Figure 8). The number of fibroblasts increased with stimulation frequency and was significantly higher in the 4 Hz stimulated group compared to the unstimulated group (Figure 8). Accordingly, levels of the MMPs and TIMP-1 were normalized to the number of cells in each well. MMP-2, MMP-9, and MT1-MMP levels at 4 Hz were lower than values obtained from fibroblasts that were left unstimulated (Figure 8). TIMP-1 levels normalized to cell number were higher at 4 Hz compared to that recorded in unstimulated fibroblasts (Figure 8).

Figure 8
TOP PANELS: Following 24 hours of electrical stimulation, LV myocardial fibroblasts retained morphological characteristics (Scale bar: 20 μm). The wells stimulated at 4 Hz contained a larger number of FIBROs than wells that were left unstimulated ...

DISCUSSION

A proteolytic event that contributes to adverse left ventricular (LV) remodeling following myocardial infarction (MI) is changes in the abundance and balance between the matrix metalloproteinases (MMPs) and the endogenous tissue inhibitors of the MMPs (TIMPs). Accordingly, the present study determined whether electrical stimulation using low-amplitude, high-frequency pulses (LHFS) within a formed MI scar would alter the course of regional MI remodeling. Specifically, in the present study LHFS was initiated in the MI scar at 21 days post-MI and maintained for the final 7 days of the study period. In addition, the effects of electrically stimulating isolated fibroblasts using high frequency (4Hz) pulses on MMP/TIMP release were examined. The main findings of this study were three-fold. First, LHFS attenuated MI thinning and increased regional stiffness of the MI region. Second, LHFS reduced the levels of certain MMP subtypes and increased TIMP-1 levels, which was accompanied by increased collagen content in the MI region. Finally, electrical stimulation of myocardial fibroblasts resulted in a decrease in the release of specific MMP types and an increase in TIMP-1 levels; suggesting that the in vivo effects with respect to the decrease in interstitial MMP activity may have been due to direct effects of electrical stimulation on fibroblasts/myofibroblasts that populate the MI region. Taken together, these findings provide evidence that a targeted non-pharmacological approach through subthreshold electrical stimulation of the MI region may represent a novel means to attenuate and/or even prevent adverse LV remodeling post-MI.

LV remodeling post-MI is characterized by changes in the cellular and extracellular constituents at the infarcted region.2, 3 The present study utilized a porcine model of MI that has been characterized in terms of infarct expansion, regional changes in MI geometry, as well as MMP and TIMP levels.4, 5 Specifically, in this clinically relevant MI model, permanent occlusion of the obtuse marginals of the circumflex artery results in an MI size of 21% and is associated with progressive LV dilation and thinning of the MI region (termed as infarct expansion).4 The rationale for selecting 21 days post-MI as the time point at which LHFS was initiated in the present study was two-fold: First, in rodent MI models, interference with the early the post-MI response in terms of modifying the cellular and/or extracellular characteristics of the MI region has been associated with deleterious effects, such as an increased incidence of LV rupture.24 Second, during the later stages of MI remodeling, cellular and extracellular events that occur within the MI region culminate in the formation of a fibrotic scar (review3). The presence of a fibrotic scar, which is generally considered to be non-conductive, was used to advantage in the present study with respect to confining the electrical propagation of LHFS to within the MI region, as evidenced in ECG recordings. Therefore, initiating LHFS at 21 days post-MI in this proof-of-concept study avoided the confounding influences with respect to interrupting the acute MI “healing” response as well as provide sufficient time for the development of a non-conductive substrate. Nevertheless, it must be recognized that sufficient remodeling of the MI region with respect to fibrotic deposition could occur earlier than 21 days, and thereby, may provide a suitable substrate to initiate LHFS earlier in the course of the post-MI remodeling process.

In the present study, LHFS over the final 7 days of the study period prevented the progressive thinning of the MI region. The presence of a thinned MI region results in a heterogeneous distribution of LV stress and strain patterns,25 which may place the viable, remote myocardium at a mechanical disadvantage and lead to a downward spiral of further MI expansion.3 Using mathematical stimulation, Pilla et al. reported that increased stiffness in the MI region was predictive of an attenuation of LV dilation post-MI.6 In the present study, and consistent with past reports, myocardial stiffness at the MI region was differentially increased with LHFS and associated with the increase in collagen content in the MI region. Moreover, LHFS instituted during the final week of the post-MI study period attenuated the progression of LV dilation. However, despite this attenuation, the LV remodeling process was not “reversed” in that LV end-diastolic volumes were similar between the two MI groups. A potential explanation for this finding is that the duration of LHFS was too short for the effects on regional LV geometry to be translated into effects on global LV dilation. Future studies in which LHFS is instituted earlier and/or maintained for a longer duration post-MI would be required to further characterize the temporal effects of LHFS with respect to LV remodeling post-MI.

The MMPs contribute to proteolysis and remodeling of the extracellular matrix (ECM) and a causal role for several MMP types in LV remodeling post-MI has been described.2, 24, 26 Consistent with past findings, MMP-2 and MMP-9 levels were increased within the MI region, but MMP-2 levels were reduced and there was a trend towards lower MMP-1 levels with LHFS. Concomitantly, there was an inverse relationship between MMP-1 levels and collagen content. TIMP-1 levels within the MI region, however, were higher with LHFS. While changes in the relative ex vivo abundance of MMP types is an important consideration with respect to in vivo proteolytic activity, it must be recognized that processing of the samples for assay can result in dissociation of MMP complexes with TIMPs and other interstitial proteins as well as linearization of the pro- and active forms of the MMPs. In order to avoid these inherent limitations of ex vivo approaches, the present study utilized a previously validated small peptide substrate with a quenched fluorescent moiety, which when cleaved by MMPs, would yield a detectable fluorescent signal.17 Results from this analysis showed that in vivo interstitial MMP activity within the MI region was reduced with LHFS. However, it must be recognized that the fluorogenic substrate used in the present study provided an “integrative” index of contributions from several MMP types. This laboratory has recently demonstrated that MMP substrates with a more specific amino acid sequence, and thereby imparting greater MMP specificity, can be successfully utilized with this microdialysis system.17, 27 Thus, future studies that utilize different MMP substrates would allow identification of the effects of LHFS on the activity of specific MMP types within the MI region. Nevertheless, findings from the present study demonstrated that the ex vivo reductions in the levels of certain MMPs with a concomitant increase in TIMP-1 levels likely contributed to the in vivo reduction of interstitial MMP activity, which in turn, suggests that there was a net in vivo reduction in ECM proteolysis that contributed to the increase in collagen content of the MI region with LHFS.

MMPs and TIMPs are synthesized and released by a number of myocardial cell types, including myocytes and fibroblasts.18, 28, 29 In addition, inflammatory cell types, such as macrophages and neutrophils, and myofibroblasts, which are differentiated fibroblasts also release MMPs.18 While myocytes comprise a majority of the cellular volume of the viable myocardium, there are relatively few myocytes – if any – within the MI region.1, 2 Indeed, in the present study, there was an absence of immunostaining for α-sarcomeric actin within the MI region. Therefore, it is unlikely that myocytes were a source for the LHFS-related changes in MMP/TIMP levels within the MI region. Furthermore, myeloperoxidase activity levels were similar to non-MI control values in the remote and MI regions of pigs with or without LHFS. This finding suggests that by 28 days post-MI, the effects of LHFS on MMP/TIMP levels were unlikely to be driven by modulating inflammation. Nevertheless, immunohistochemical staining of the MI region revealed that TIMP-1 colocalized with cells that stained for α-smooth muscle actin, a well-established marker for myofibroblasts.18 However, the colocalization of TIMP-1 to cells staining for α-smooth muscle actin appeared to be greater with LHFS. Moreover, the levels of cell markers for mesenchymal cell types, such as α-smooth muscle actin and vimentin, within the MI region were differentially higher with LHFS. Finally, in vitro electrical stimulation of fibroblasts reduced the abundance of MMP-2, MMP-9, and MT1-MMP when compared to fibroblasts that were left unstimulated. Concomitantly, TIMP-1 levels were increased in the fibroblasts that were subjected to in vitro electrical stimulation. Therefore, the stoichiometric balance between MMPs and TIMPs that were released from fibroblasts in response to electrical stimulation was shifted to one that would likely favor collagen accumulation. Taken together, these findings of the present study suggest that the cellular source for the differentially higher TIMP-1 levels and collagen content with LHFS were likely fibroblasts and/or myofibroblasts.

Increased levels of transforming growth factor-β (TGF-β) and activation of the TGF-β signaling pathway, which includes the TGF-β receptors R1 and R2 as well as the SMAD second messenger system, are associated in fibrosis with MI.30-33 Consistent with these past findings, the present study demonstrated that levels of TGF-β, TGF-β R1, SMAD-2/3, and phosphorylated SMAD-2/3 were higher within the MI region than control levels. Moreover, the levels of TGF-β R1 and the ratio of phosphorylated SMAD-2/3 to total SMAD-2/3 were differentially higher with LHFS. SMAD-2/3 requires phosphorylation to be chaperoned across the nuclear membrane to effect transcription of a number of genes.19, 32, 33 Fibroblasts and myofibroblasts have been previously shown to respond to TGF-β stimulation through increased synthesis and release of fibrillar collagens.1, 28, 31, 33, 34 Therefore, the findings of the present study suggest that a fundamental mechanism – at least in part – for the differentially higher collagen content within the MI region with LHFS was activation of the TGF-β signaling pathway and release of fibrillar collagen from fibroblasts / myofibroblasts.

Clinical considerations

A major consideration for selecting the LHFS parameters used in the present study was to prevent “escape” of the introduced stimuli into viable myocardium, which may have resulted in ventricular tachycardia. This is an important consideration since the presence of a fibrotic post-MI scar can form an area of conduction block35 and origination sites for reentrant rhythms due to changes in the refractory properties and/or changes in the length of conduction pathways.36 Moreover, variability in MI sizes, the presence of alternate high resistance conduction (e.g. through islets of remnant viable myocardium in the MI region), and case-to-case variation in fibrillation thresholds comprise some of the factors that must be considered in future studies that implement LHFS in the setting of MI.

The present study provided proof-of-concept that LHFS could be designed as a “therapy” to provide a targeted, non-pharmacological approach to attenuate infarct expansion after the formation of a fibrotic scar. Potential advantages of the LHFS approach include regional and temporal specificity in terms of directing treatment. For example, regional specificity would be achieved through the placement of the electrode in the area of targeted intervention, which may hold therapeutic benefit over systemic pharmacological approaches. With respect to temporal specificity, LHFS may afford the opportunity to activate and deactivate the pacemaker based up on achieving a potential remodeling response. While the prospects of implementing LHFS in the clinical setting are provocative, it must be recognized that the findings reported here, albeit determined in a clinically relevant animal model, represent just the short term effects of LHFS. For instance, it is possible that chronic LHFS could result in late arrhythmias, which would not be apparent in the current study design. Therefore, extrapolation to the clinical context must be undertaken with caution and only after further characterization of the later effects of LHFS on LV geometry and associated changes in myocardial material properties.

POTENTIAL CLINICAL IMPACT

Adverse left ventricular (LV) remodeling following myocardial infarction (MI) remains an important cause of morbidity and mortality. Current pharmacological strategies fail to interrupt or reverse the inexorable progress of post-MI remodeling. Recent translational or early clinical research has focused upon the development of new pharmacological modalities or the feasibility of delivery of exogenous cell types to prevent post-MI remodeling. However, whether endogenous cell types can be recruited and/or targeted to prevent, or even reverse, LV remodeling post-MI has not been explored. In the present proof-of-concept study, low amplitude, high frequency electrical stimulation instituted within a formed MI scar arrested the progressive thinning of the MI region and attenuated LV dilation. A likely mechanism for these findings was direct effects of electrical stimulation of fibroblasts / myofibroblasts resident within the MI region. These findings suggest that targeted electrical stimulation of the MI region can alter the endogenous substrate for post-MI remodeling. In light of the fact that multisite myocardial pacing is a commonly utilized clinical tool, then translational studies to extend the basic observations of the present study are warranted.

Supplementary Material

tables

Acknowledgments

FUNDING SOURCES

This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-45024, HL-97012, and PO1-48788, and a VA Merit Award (FGS)

Footnotes

DISCLOSURES

Rupak Mukherjee: No relationship to disclose

William T. Rivers: No relationship to disclose

Jean Marie Ruddy: No relationship to disclose

Robert G. Matthews: No relationship to disclose

Christine N. Koval: No relationship to disclose

Rebecca A. Plyler: No relationship to disclose

Eileen I. Chang: No relationship to disclose

Risha K. Patel: No relationship to disclose

Christine B. Kern: No relationship to disclose

Robert E. Stroud: No relationship to disclose

Francis G. Spinale: Grant recipient from NIH and the Veteran's Administration (VA)

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

1. Cleutjens JP, Creemers EE. Integration of concepts: cardiac extracellular matrix remodeling after myocardial infarction. J Card Fail. 2002;8(6 Suppl):S344–348. [PubMed]
2. Creemers EE, Cleutjens JP, Smits JF, Daemen MJ. Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure? Circ Res. 2001;89(3):201–210. [PubMed]
3. Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation. 2000;101(25):2981–2988. [PubMed]
4. Mukherjee R, Brinsa TA, Dowdy KB, Scott AA, Baskin JM, Deschamps AM, Lowry AS, Escobar GP, Lucas DG, Yarbrough WM, Zile MR, Spinale FG. Myocardial infarct expansion and matrix metalloproteinase inhibition. Circulation. 2003;107(4):618–625. [PubMed]
5. Yarbrough WM, Mukherjee R, Brinsa TA, Dowdy KB, Scott AA, Escobar GP, Joffs C, Lucas DG, Crawford FA, Jr., Spinale FG. Matrix metalloproteinase inhibition modifies left ventricular remodeling after myocardial infarction in pigs. J Thorac Cardiovasc Surg. 2003;125(3):602–610. [PubMed]
6. Pilla JJ, Gorman JH, 3rd, Gorman RC. Theoretic impact of infarct compliance on left ventricular function. Ann Thorac Surg. 2009;87(3):803–810. [PMC free article] [PubMed]
7. Wilson EM, Moainie SL, Baskin JM, Lowry AS, Deschamps AM, Mukherjee R, Guy TS, John-Sutton MG, Gorman JH, 3rd, Edmunds LH, Jr., Gorman RC, Spinale FG. Region- and type-specific induction of matrix metalloproteinases in post-myocardial infarction remodeling. Circulation. 2003;107(22):2857–2863. [PubMed]
8. Brighton CT, Wang W, Clark CC. The effect of electrical fields on gene and protein expression in human osteoarthritic cartilage explants. J Bone Joint Surg Am. 2008;90(4):833–848. [PubMed]
9. Assimacopoulos D. Wound healing promotion by the use of negative electric current. Am Surg. 1968;34(6):423–431. [PubMed]
10. Kloth LC. How to use electrical stimulation for wound healing. Nursing. 2002;32(12):17. [PubMed]
11. Kloth LC. Electrical stimulation for wound healing: a review of evidence from in vitro studies, animal experiments, and clinical trials. Int J Low Extrem Wounds. 2005;4(1):23–44. [PubMed]
12. Shi G, Rouabhia M, Meng S, Zhang Z. Electrical stimulation enhances viability of human cutaneous fibroblasts on conductive biodegradable substrates. J Biomed Mater Res A. 2008;84(4):1026–1037. [PubMed]
13. Sun S, Wise J, Cho M. Human fibroblast migration in three-dimensional collagen gel in response to noninvasive electrical stimulus. I. Characterization of induced three-dimensional cell movement. Tissue Eng. 2004;10(9-10):1548–1557. [PubMed]
14. Cho MR, Marler JP, Thatte HS, Golan DE. Control of calcium entry in human fibroblasts by frequency-dependent electrical stimulation. Front Biosci. 2002;7:a1–8. [PubMed]
15. Kamrin BB. Induced collagenolytic activity by electrical stimulation of embryonic fibroblasts in tissue culture. J Dent Res. 1974;53(6):1475–1483. [PubMed]
16. Schiller NB, Shah PM, Crawford M, DeMaria A, Devereux R, Feigenbaum H, Gutgesell H, Reichek N, Sahn D, Schnittger I, Silverman NH, Tajik AJ. Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms. J Am Soc Echocardiogr. 1989;2(5):358–367. [PubMed]
17. Deschamps AM, Yarbrough WM, Squires CE, Allen RA, McClister DM, Dowdy KB, McLean JE, Mingoia JT, Sample JA, Mukherjee R, Spinale FG. Trafficking of the membrane type-1 matrix metalloproteinase in ischemia and reperfusion: relation to interstitial membrane type-1 matrix metalloproteinase activity. Circulation. 2005;111(9):1166–1174. [PubMed]
18. Mukherjee R, Mingoia JT, Bruce JA, Austin JS, Stroud RE, Escobar GP, McClister DM, Jr., Allen CM, Alfonso-Jaume MA, Fini ME, Lovett DH, Spinale FG. Selective spatiotemporal induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 transcription after myocardial infarction. Am J Physiol Heart Circ Physiol. 2006;291(51):H2216–2228. [PubMed]
19. Jones JA, Barbour JR, Stroud RE, Bouges S, Stephens SL, Spinale FG, Ikonomidis JS. Altered transforming growth factor-beta signaling in a murine model of thoracic aortic aneurysm. J Vasc Res. 2008;45(6):457–468. [PMC free article] [PubMed]
20. Flack EC, Lindsey ML, Squires CE, Kaplan BS, Stroud RE, Clark LL, Escobar PG, Yarbrough WM, Spinale FG. Alterations in cultured myocardial fibroblast function following the development of left ventricular failure. J Mol Cell Cardiol. 2006;40(4):474–483. [PubMed]
21. Squires CE, Escobar GP, Payne JF, Leonardi RA, Goshorn DK, Sheats NJ, Mains IM, Mingoia JT, Flack EC, Lindsey ML. Altered fibroblast function following myocardial infarction. J Mol Cell Cardiol. 2005;39(4):699–707. [PubMed]
22. Kato S, Ivester CT, Cooper Gt, Zile MR, McDermott PJ. Growth effects of electrically stimulated contraction on adult feline cardiocytes in primary culture. Am J Physiol. 1995;268(6 Pt 2):H2495–2504. [PubMed]
23. Rivest L-P. Statistical properties of winsorized means for skewed distributions. Biometrika. 1994;81(2):373–383.
24. Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, Dyspersin GD, Cleutjens JP, Shipley M, Angellilo A, Levi M, Nube O, Baker A, Keshet E, Lupu F, Herbert JM, Smits JF, Shapiro SD, Baes M, Borgers M, Collen D, Daemen MJ, Carmeliet P. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med. 1999;5(10):1135–1142. [PubMed]
25. Pilla JJ, Blom AS, Gorman JH, 3rd, Brockman DJ, Affuso J, Parish LM, Sakamoto H, Jackson BM, Acker MA, Gorman RC. Early postinfarction ventricular restraint improves borderzone wall thickening dynamics during remodeling. Ann Thorac Surg. 2005;80(6):2257–2262. [PubMed]
26. Creemers E, Cleutjens J, Smits J, Heymans S, Moons L, Collen D, Daemen M, Carmeliet P. Disruption of the plasminogen gene in mice abolishes wound healing after myocardial infarction. Am J Pathol. 2000;156(6):1865–1873. [PMC free article] [PubMed]
27. Deschamps AM, Zavadzkas J, Murphy RL, Koval CN, McLean JE, Jeffords L, Saunders SM, Sheats NJ, Stroud RE, Spinale FG. Interruption of endothelin signaling modifies membrane type 1 matrix metalloproteinase activity during ischemia and reperfusion. Am J Physiol Heart Circ Physiol. 2008;294(2):H875–883. [PMC free article] [PubMed]
28. Spinale FG. Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function. Physiol Rev. 2007;87(4):1285–1342. [PubMed]
29. Porter KE, Turner NA. Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol Ther. 2009;123(2):255–278. [PubMed]
30. Ellmers LJ, Scott NJ, Medicherla S, Pilbrow AP, Bridgman PG, Yandle TG, Richards AM, Protter AA, Cameron VA. Transforming growth factor-beta blockade down-regulates the renin-angiotensin system and modifies cardiac remodeling after myocardial infarction. Endocrinology. 2008;149(11):5828–5834. [PubMed]
31. Jugdutt BI. Extracellular matrix and cardiac remodeling. In: Villarreal FJ, editor. Interstitial fibrosis in heart failure. Springer; New York: 2000. pp. 23–56.
32. Bujak M, Ren G, Kweon HJ, Dobaczewski M, Reddy A, Taffet G, Wang XF, Frangogiannis NG. Essential role of Smad3 in infarct healing and in the pathogenesis of cardiac remodeling. Circulation. 2007;116(19):2127–2138. [PubMed]
33. Bujak M, Frangogiannis NG. The role of TGF-beta signaling in myocardial infarction and cardiac remodeling. Cardiovasc Res. 2007;74(2):184–195. [PMC free article] [PubMed]
34. Sun Y, Weber KT. Infarct scar: a dynamic tissue. Cardiovasc Res. 2000;46(2):250–256. [PubMed]
35. Soejima K, Stevenson WG, Maisel WH, Sapp JL, Epstein LM. Electrically unexcitable scar mapping based on pacing threshold for identification of the reentry circuit isthmus: feasibility for guiding ventricular tachycardia ablation. Circulation. 2002;106(13):1678–1683. [PubMed]
36. Stevenson WG, Khan H, Sager P, Saxon LA, Middlekauff HR, Natterson PD, Wiener I. Identification of reentry circuit sites during catheter mapping and radiofrequency ablation of ventricular tachycardia late after myocardial infarction. Circulation. 1993;88(4 Pt 1):1647–1670. [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • EST
    EST
    Published EST sequences
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...