Alternative and innovative targeted strategies hold relevance in improving the current treatments for ischemic heart disease (IHD). One potential treatment modality, gene targeting, may provide a unique alternative to current IHD therapies. The principal function of gene targeting in IHD is to augment the expression of an endogenous gene through amplification of an exogenous gene, delivered by a plasmid or a viral vector to enhance myocardial perfusion, and limit the long-term sequelae. The initial clinical studies of gene targeting in IHD were focused upon induction of angiogenic factors and the outcomes were equivocal. Nevertheless, significant advancements have been made in viral vectors, mode of delivery, and potentially relevant targets for IHD. Several of these advancements, particularly with a focus on translational large animal studies, are the focus of this review. The development of novel vectors with prolonged transduction efficiency and minimal inflammation, coupled with hybrid perfusion-mapping delivery devices, and improving the safety of vector use and efficacy of gene systems are but a few of the exciting progresses that are likely to proceed to clinical studies in the near future.
gene therapy; myocardial infarction; ischemia; plasmid; adenovirus; adeno-associated virus
Membrane type 1 matrix metalloproteinase (MT1-MMP) is critical to a number of proteolytic and profibrotic events. However, upstream regulation of MT1-MMP with myocardial ischemia–reperfusion remains poorly understood. MicroRNAs regulate post-transcriptional events, and in silico mapping has identified a conserved sequence in MT1-MMP for microRNA-133a. This study tested the hypothesis that changes in microRNA-133a regulation occur with myocardial ischemia–reperfusion, which contributes to time- and region-dependent changes in MT1-MMP activity and processing of MT1-MMP substrates.
Yorkshire pigs (n = 12) underwent ischemia–reperfusion (90 minutes ischemia and 120 minutes reperfusion), where regional preload recruitable stroke work (sonomicrometry), interstitial MT1-MMP activity (microdialysis), Smad2 abundance (immunoblotting), and interstitial microRNA-133a (polymerase chain reaction) were determined within the ischemia–reperfusion and remote regions. Human left ventricular fibroblasts were transduced with microRNA-133a and anti–microRNA-133a (lentivirus) to determine the effects on MT1-MMP protein abundance.
With ischemia–reperfusion, regional preload recruitable stroke work decreased from steady state (139 ± 20 mm Hg to 44 ± 11 mm Hg, P <.05) within the ischemia–reperfusion region. MT1-MMP activity increased in both regions. Phosphorylated Smad2 increased within the ischemia–reperfusion region. Both in vitro and in vivo interstitial levels of microRNA-133a decreased with ischemia and returned to steady-state levels with reperfusion. In vitro transduction of microRNA-133a in left ventricular fibroblasts decreased MT1-MMP levels.
Modulation of MT1-MMP activity and microRNA-133a exportation into the myocardial interstitium occurred in the setting of acute myocardial ischemia–reperfusion. In addition, changes in microRNA-133a expression in left ventricular fibroblasts resulted in an inverse modulation of MT1-MMP abundance. Therefore, targeting of microRNA-133a represents a potentially novel means for regulating the cascade of profibrotic events after ischemia–reperfusion.
The myocardial interstitium is highly organized and orchestrated whereby small disruptions in composition, spatial relationships, or content lead to altered myocardial systolic and/or diastolic performance. These changes in extracellular matrix structure and function are important in the progression to heart failure in pressure overload hypertrophy (POH), dilated cardiomyopathy (DCM), and ischemic heart disease. The myocardial interstitium is not a passive entity, but rather a complex and dynamic microenvironment which represents an important structural and signaling system within the myocardium.
interstitium; extracellular matrix; heart failure; pressure overload hypertrophy; dilated cardiomyopathy; myocardial infarction
Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac-MLCK (cMLCK); yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined.
Methods and Results
We found that pressure-overload induced by transaortic constriction in wildtype mice reduced phosphorylated-MLC2v levels by ~40% and cMLCK levels by ~85%. To examine how a reduction in cMLCK and the corresponding reduction in pMLC2v affect function, we generated Mylk3 gene-targeted mice as well as transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure-overload led to severe heart failure in cMLCK knockout mice, but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure-overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems.
Our results suggest the novel idea that accelerated cMLCK-protein turnover by the ubiquitin-proteasome system underlie the transition from compensated hypertrophy to decompensated heart failure due to reduced phosphorylation of MLC2v.
myosin light chain; phosphorylation; heart failure
Myocyte death occurs by necrosis and caspase-mediated apoptosis in myocardial infarction (MI). In-vitro studies suggest caspase activation causes myocardial contractile protein degradation without inducing apoptosis. Thus, caspase activation may evoke left ventricular (LV) remodeling through independent processes post-MI. The effects of caspase activation on LV geometry post-MI remain unclear. This project applied pharmacologic caspase inhibition (CASPI) to a porcine model of MI.
Methods and Results
Pigs (34kg) were instrumented in order to induce 60min of coronary artery occlusion followed by reperfusion and a 7-day follow-up period. Upon reperfusion, the pigs were randomized to saline (n=12) or CASPI (n=10, IDN6734, 6mg/kg IV, then 6mg/kg/hr for 24hrs). Plasma troponin-I values were reduced with CASPI compared to saline at 24hrs post-MI (133±15 vs 189±20ng/mL, respectively, p<0.05). LV end-diastolic area (echocardiography) and inter-regional length (sonomicrometry) increased from baseline in both groups but were attenuated with CASPI by 40% and 90%, respectively (p<0.05). Myocyte length was reduced with CASPI compared to saline (128±3 vs 141±4μm, respectively, p<0.05). Plasma free-pro-MMP-2 values increased from baseline with CASPI (27±6%, p<0.05) indicative of reduced conversion to active MMP-2. Separate in-vitro studies demonstrated that activated caspase species cleaved pro-MMP-2 yielding active MMP-2 forms and that MMP activity was increased in the presence of activated caspase-3.
CASPI attenuated regional and global LV remodeling post-MI and altered viable myocyte geometry. Caspases increased MMP activity in-vitro while CASPI modified conversion of MMP-2 to the active form in-vivo. Taken together, the results of the present study suggest that the elaboration of caspases post-MI likely contribute to LV remodeling through both cellular and extracellular mechanisms.
myocardial infarction; remodeling; caspase inhibition; left ventricular
Background and Objective
A robust release of endothelin-1-1 (ET) with subsequent ETA subtype receptor (ET-AR) activation occurs in patients following cardiac surgery requiring cardiopulmonary bypass (CPB). Increased ET-AR activation has been identified in patients with poor LV function (reduced ejection fraction; EF). Accordingly, this study tested the hypothesis that a selective ET-AR antagonist (ET-ARA) administered peri-operatively would favorably affect post-CPB hemodynamic profiles in patients with a pre-existing poor LVEF.
Methods and Results
Patients (n=29; 66±2 yrs) with a reduced LVEF (37±2%) were prospectively randomized, in a blinded fashion, at the time of elective coronary revascularization and/or valve replacement requiring CPB, to infusion of the highly-selective and potent ET-ARA, sitaxsentan at 1 or 2 mg/kg (IV bolus; n=9, 10 respectively) or vehicle (saline; n=10). Infusion of the ET-ARA/vehicle was performed immediately prior to separation from CPB and again at 12 hrs post-CPB. ET and hemodynamic measurements were performed at baseline, at separation from CPB (Time 0) and at 0.5, 6, 12, 24 hrs post-CPB. Baseline plasma ET (4.0±0.3 fmol/mL) was identical across all 3 groups, but when compared to pre-operative, baseline values obtained from age matched subjects with a normal LVEF (n=37;LVEF>50%), were significantly increased (2.9±0.2 fmol/mL, p<0.05) Baseline systemic (SVR; 1358±83 d·s·cm-5) and pulmonary (PVR; 180±23 d·s·cm-5) vascular resistance were equivalent in all 3 groups. As a function of Time 0, SVR changed in an equivalent fashion in the post-CPB period, but a significant ET-ARA effect was observed for PVR (ANOVA; p<0.05). For example at 24 hrs post-CPB, PVR increased by 40 d.scm-5 in the vehicle group, but directionally decreased by over 40 d·s·cm-5 in the 2 mg/kg ETARA group (p<0.05). Total adverse events were equivalently distributed across the ET-ARA/placebo groups.
These unique findings demonstrated that infusion of an ET-ARA in high risk cardiac surgery patients was not associated with significant hemodynamic compromise. Moreover, ET-ARA favorably affected PVR in the early post-operative period. Thus, the ET-AR serves as a potential pharmacological target for improving outcomes following cardiac surgery in patients with compromised LV function.
Endothelin-1; receptor antagonist; cardiac surgery; systolic dysfunction
Despite advances in reperfusion therapy, acute coronary syndromes can still result in myocardial injury and subsequent MI. Molecular, cellular, and interstitial events antecedent to the acute MI culminate in deleterious changes in the size, shape, and function of the left ventricle (LV), collectively termed LV remodeling. Three distinct anatomical and physiologic LV regions can be described post-MI: the infarct, borderzone, and remote regions. Given the complexity of post-MI remodeling, imaging modalities must be equally diverse to elucidate this process. The focus of this review will first be upon cardiovascular magnetic resonance imaging (MRI) of the anatomical and pathophysiological LV regions of greatest interest with regard to the natural history of the post-MI remodeling process. This review will examine imaging modalities which provide translational and molecular insight into burgeoning treatment fields for the attenuation of post-MI remodeling, such as cardiac restraint devices and stem cell therapy.
myocardial infarction; remodeling; plasma profiling
The direct consequences of a persistently increased myocardial expression of the unique matrix metalloproteinase (MMP), membrane type-1 (MT1-MMP) on myocardial remodeling remained unexplored.
Cardiac restricted MT1-MMPexp was constructed in mice using the full length human MT1-MMP gene ligated to the myosin heavy chain promoter, which yielded approximately a 200% increase in MT1-MMP when compared to age/strain matched wild type mice (WT). LV function and geometry was assessed by echocardiography in 3 month (“young”) WT (n=32) and MT1-MMPexp (n=20) mice, and compared to 14 month (“middle age”) WT (n=58) and MT1-MMPexp (n=35) mice. LV end-diastolic volume was similar between the WT and MT1-MMPexp young groups as was LV ejection fraction. In the middle age WT mice, LV end-diastolic volume and ejection fraction was similar to young WT mice. However, in the MT1-MMPexp middle age mice, LV end-diastolic volume was approximately 43% higher and LV ejection fraction 40% lower (both p<0.05). Moreover, in the middle age MT1-MMPexp mice, myocardial fibrillar collagen increased by nearly 2-fold and was associated with an approximate 3-fold increase in the processing of the pro-fibrotic molecule, latency-associated transforming growth factor binding protein. In a second study, 14 day survival following myocardial infarction was significantly lower in middle aged MT1-MMPexp mice.
Persistently increased myocardial MT1-MMP expression, in and of itself, caused LV remodeling, myocardial fibrosis, dysfunction and reduced survival following myocardial injury. These findings suggest that MT1-MMP plays a mechanistic role in adverse remodeling within the myocardium.
matrix; myocardial remodeling; ventricular function; aging
Epsilon aminocaproic acid (EACA) is used in cardiac surgery to modulate plasmin activity (PLact). The present study developed a fluorogenic-microdialysis system to measure in-vivo region specific temporal changes in PLact following EACA administration.
Pigs (25-35kg) received EACA (75mg/kg, n=7) or saline in which microdialysis probes were placed in the liver, myocardium, kidney and quadricep muscle. The microdialysate contained a plasmin specific fluorogenic peptide and fluorescence emission, which directly reflected PLact, determined at baseline, 30, 60, 90 and 120 minutes following EACA/vehicle infusion.
EACA caused significant decreases in liver and quadricep PLact at 60, 90, 120 minutes and at 30, 60, 120 minutes respectively (p<0.05). In contrast, EACA induced significant biphasic changes in heart and kidney PLact profiles with initial increases followed by decreases at 90 and 120 minutes (p<0.05). The peak EACA interstitial concentrations for all compartments occurred at 30 minutes post infusion, and were 5-fold higher in the renal compartment and 4-fold higher in the myocardium, when compared to the liver or muscle (p<0.05).
Using a large animal model and in-vivo microdialysis measurements of plasmin activity, the unique findings from this study were 2-fold. First, EACA induced temporally distinct plasmin activity profiles within the plasma and interstitial compartments. Second, EACA caused region specific changes in plasmin activity profiles. These temporal and regional heterogeneic effects of EACA may have important therapeutic considerations when managing fibrinolysis in the perioperative period.
Congestive heart failure (HF) is a clinical syndrome, with hallmarks of fatigue and dyspnea, which continues to be highly prevalent and morbid. Due to the growing burden of HF as the population ages, the need to develop new pharmacologic treatments and therapeutic interventions is of paramount importance. Common pathophysiologic features of HF include changes in left ventricle (LV) structure, function, and neurohormonal activation. The recapitulation of the HF phenotype in large animal models can allow for the translation of basic science discoveries into clinical therapies. Models of myocardial infarction/ischemia, ischemic cardiomyopathy, ventricular pressure and volume overload, and pacing induced dilated cardiomyopathy have been created in dogs, pigs, and sheep for the investigation of HF and potential therapies. Large animal models recapitulating the clinical HF phenotype and translating basic science to clinical applications have successfully traveled the journey from bench to bedside. Undoubtedly, large animal models of HF will continue to play a crucial role in the elucidation of biologic pathways involved in HF and the development and refinement of HF therapies.
Myocardial infarction; myocardial remodeling; overload states; rapid pacing
Basic studies have suggested that cross-talk exists between the endothelin-A receptor (ET-AR) and tumor necrosis factor signaling pathway. This study tested the hypothesis that administration of an ET-AR antagonist at the separation from cardiopulmonary bypass would alter the tumor necrosis factor activation in the early post operative period.
Patients (n = 44) were randomized to receive bolus infusion of vehicle, 0.1, 0.5, 1, or 2 mg/kg of the ET-AR antagonist (sitaxsentan) at the separation from cardiopulmonary bypass (n=9, 9, 9, 9, and 8 respectively). Plasma levels of tumor necrosis factor-α and soluble tumor necrosis factor receptor 1 and 2 were measured.
Compared to the vehicle group at 24 hours, plasma levels of tumor necrosis factor-α and tumor necrosis factor receptor 2 (indicative of receptor activation) were reduced in the 1 mg/kg ET-AR antagonist group (by ~13 pg/mL and ~0.5 ng/mL respectively; p<0.05). Plasma tumor necrosis factor receptor I levels also decreased (by ~1 ng/mL) following infusion of the higher doses of the ET-AR antagonist and remained lower (by ~3 ng/mL) at 24 hours post infusion (p<0.05). In addition, a dose effect was observed between the ET-AR antagonist and these indices of tumor necrosis factor activation (p<0.01).
This study demonstrated a mechanistic relationship between the ET-AR and tumor necrosis factor receptor activation in the post cardiac surgery period. Thus, in addition to the potential cardiovascular effects, a selective ET-AR antagonist can modify other biological processes relevant to the post cardiac surgery setting. (word count:245/250)
Cardiopulmonary Bypass (CPB); Inflammation; Cytokines
Past studies have clearly established that matrix metalloproteinases (MMPs) contribute to adverse myocardial remodeling with ischemia and reperfusion. However, these studies measured MMP levels in extracted samples, and therefore whether and to what degree actual changes in interstitial MMP activity occur within the human myocardium in the context of ischemia/reperfusion remained unknown.
The present study directly quantified MMP interstitial activity (MMPact) within the myocardium of patients (n=14) undergoing elective cardiac surgery during steady-state conditions, as well as during and following an obligatory period of myocardial arrest and reperfusion achieved by cardiopulmonary bypass (CPB). Interstitial MMP activity was continuously monitored using a validated MMP fluorogenic substrate, a microdialysis system placed within the myocardium, and in-line fluorescent detection system.
MMP activity, as measured by fluorescent emission, reached a stable steady state level by 10 minutes following deployment of the microdialysis system. During initiation of CPB, MMP activity increased by 20% from baseline values (p<0.05), and then rapidly fell with cardiac arrest and longer periods of CPB. However, with restoration of myocardial blood flow and separation from CPB, MMP interstitial activity increased by over 30% from baseline (p<0.05).
The present study directly demonstrated that MMP proteolytic activity exists within the human myocardial interstitium and is a dynamic process under conditions such as myocardial arrest and reperfusion.
microdialysis; cardiac surgery; fluorogenic substrates; extracellular matrix
Aortic valve stenosis is a common cause of left ventricular pressure overload, a pathologic process that elicits myocyte hypertrophy and alterations in extracellular matrix composition, both of which contribute to increases in left ventricular stiffness. However, clinical and animal studies suggest that increased myocardial extracellular matrix fibrillar collagen content occurs later in the time course of left ventricular pressure overload at a time coincident with severe abnormalities in diastolic function followed by the development of symptomatic heart failure. Aortic valve replacement remains the most effective treatment for elimination of chronic pressure overload secondary to aortic stenosis but has traditionally been recommended only after the onset of clinical symptoms. However, long-term follow-up of symptomatic aortic stenosis patients after aortic valve replacement suggests that valve replacement may not result in complete reversal of the maladaptive changes that occur within the myocardial extracellular matrix secondary to the pressure overload state. Quite the contrary, residual left ventricular extracellular matrix abnormalities such as these are likely responsible for persistent abnormalities in diastolic function and increased morbidity and mortality after aortic valve replacement. Thus, defining the mechanisms and pathways responsible for regulating the myocardial extracellular matrix during the natural history of aortic stenosis may provide a means by which to detect crucial structural milestones and thereby permit more precise identification of the development of maladaptive left ventricular remodeling.
Congenital bicuspid aortic valves (BAVs) result from fusion of two valve cusps, resulting in left-noncoronary (L-N), right-left (R-L), and right-noncoronary (R-N) morphologies. BAVs predispose to ascending thoracic aortic aneurysms (ATAAs). This study hypothesized that ATAAs with each BAV morphology group possess unique signatures of matrix metalloproteinases (MMPs) and endogenous tissue inhibitors (TIMPs).
ATAA tissue from 46 BAV patients was examined for MMP/TIMP abundance and global MMP activity compared to normal aortic specimens (n=15). Proteolytic balance was calculated as the ratio of MMP abundance to a composite TIMP score (TS). Results were stratified by valve morphology group (L-N (n=6), R-L (n=31), and R-N(n=9)).
The BAV specimens (p<0.05 vs. normal aorta, 100%) displayed elevated global MMP activity (273±63%), MMP-9 (263±47%), and decreased MMP -7 (56±10%), -8 (58±11%), TIMP -1 (63±7%) and -4 (38±3%). The R-L group showed increased global MMP activity (286±89%) and MMP-9 (267±55%) with reduced MMP -7 (45±7%) -8 (68±15%), TIMP -1 (58±7%) and -4 (35±3%). The L-N group showed elevated global MMP activity (284±71%), and decreased MMP-8 (37±17%) and TIMP-4 (48±14). In the R-N group, MMP -7 (46±13%) and -8 (36±17%), and TIMP -1 (59±10) and -4 (42±5%) were decreased. The R-L group demonstrated an increased proteolytic balance for MMP-1, MMP-9, and MMP-12 relative to L-N and R-N.
Each BAV morphology group possesses a unique signature of MMPs and TIMPs. MMP/TIMP score ratios suggest that the R-L group may be more aggressive, justifying earlier surgical intervention.
MicroRNAs (miRs) are small noncoding RNAs that recognize and bind to mRNAs and inhibit protein translation or degrade mRNA. Studies in animal models have suggested that miRs play a translational or post-translational regulatory role in myocardial growth, fibrosis, viability, and remodeling. However, whether specific temporal changes in miRs occur in patients during the LV remodeling process that follows a myocardial infarction (post-MI) remains unknown. The purpose of the current pilot study was to test the hypothesis that plasma miRs could be reliably measured in post-MI patients and that there is the relationship between temporal changes in specific miRs and post-MI LV structural remodeling.
Methods and Results
LV end-diastolic volume (EDV, echocardiography) and plasma miR were measured in age matched referent controls (CTL n=12) and post-MI patients (n=12) from day 2 through day 90 post-MI. Selected miRs (miR-1, -21, -29a, 133a, 208) were measured using quantitative rt-PCR and normalized for endogenous snRNA U6. Following MI, LVEDV increased progressively compared to CTL; this was accompanied by time dependent changes in specific miRs. For example, miR-21 initially fell 2 days post-MI (0.3±0.1 fold vs. CTL, p< 0.05), increased 5 days post-MI (2±1 fold vs. CTL, p< 0.05), and returned to CTL values at later post-MI time points. In contrast, miR-29a increased 5 days post-MI (4±1 fold vs. CTL, p< 0.05) and then fell to CTL at later time points. miR-208 increased 5 days post-MI (3±1 fold vs. CTL, p< 0.05) and remained elevated up to 90 days post-MI.
A time-dependent change in miRs occurred in post-MI patients that included an early and robust rise in miRs that have been shown to affect myocardial growth, fibrosis and viability. Thus, serially profiling miRs in the plasma of post-MI patients may hold both mechanistic and prognostic significance.
Myocardial Infarction; Remodeling; microRNA
Left ventricular (LV) remodeling following myocardial infarction (MI) is associated with increased levels of specific matrix metalloproteinases (MMPs) and relative reduction of endogenous tissue inhibitors of the MMPs (TIMPs). However, transcriptional mechanisms for the disparate post-MI MMP/TIMP expression remain unknown. Using murine constructs designed to report gene promoter activation, this study tested the hypothesis that distinctly different temporal profiles of MMP-2, MMP-9, and TIMP-1 transcription occurs post-MI.
Transcriptional activity (β-galactosidase (β-gal) reporter constructs) of MMP-2 (n=49), MMP-9 (n=62), or TIMP-1 (n=40) was assayed at 1 hour (acute), and 1 – 28 days after MI (coronary ligation) in transgenic reporter mice. At 7 days post-MI, the area of promoter activation normalized to LV area was increased from acute values for MMP-2 (63.4±5.8 vs 1.1±1.0 %, p<0.05) and MMP-9 (53.1±6.1 vs 1.3±0.9 %, p<0.05). While TIMP-1 promoter activation at 7 days post-MI increased from acute values (3.6±1.3 vs 0.3±0.5%, p<0.05), this increase was smaller than that for MMP-2 or MMP-9 (both p<0.05). MMP-2 promoter activation peaked in the MI region at 7 days post-MI and MMP-9 promoter activation was highest in the border region at 7 and 14 days post-MI. TIMP-1 promoter activation peaked within the MI region at 7 days post-MI and within the remote region at 14 days post-MI.
These findings provided direct in vivo evidence that discordant changes in temporal and spatial patterns of MMP/TIMP transcription occurs with MI. Restoration of TIMP-1 promoter activation may represent a molecular therapeutic target to attenuate/prevent adverse post-MI LV remodeling.
Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; Myocardial infarction; Remodeling
Patients with severe left ventricular (LV) pressure overload (LVPO) secondary to aortic stenosis can present with signs and symptoms of heart failure despite normal LV ejection fractions (LVEF). This process occurs, at least in part, as a result of LVPO-induced extracellular matrix (ECM) remodeling which promulgates increased LV stiffness and impaired diastolic function. However, the determinants which drive ECM remodeling in this form of LVPO remain to be fully defined.
LVPO was induced in mature pigs (n=15) by progressive ascending aortic cuff inflation (once/week/4 weeks) whereby LV mass, LVEF, and regional myocardial stiffness (rKm) were compared to referent controls (n=12). Determinants of ECM remodeling were assessed by measuring levels of mRNA expression for fibrillar collagens, matrix metalloproteinases (MMPs), and the tissue inhibitors of MMP-1 and -4 (TIMP-1, -4).
With LVPO, LV mass and rKm increased by 2- and 3-fold, respectively, compared to control, with no change in LVEF. LV myocardial collagen increased approximately 2-fold which was accompanied by reduced solubility (i.e. increased cross-linking) with LVPO, but mRNA expression for fibrillar collagen and MMPs remained relatively unchanged. In contrast, a robust increase in mRNA expression for TIMP-1 and -4 occurred with LVPO.
In a progressive model of LVPO, which recapitulates the phenotype of aortic stenosis, increased ECM accumulation and subsequently increased myocardial stiffness was not due to increased fibrillar collagen expression, but rather due to determinants of post-translational control which included increased collagen stability (thereby resistant to MMP degradation) and increased endogenous MMP inhibition. Targeting these ECM post-translational events with LVPO may hold both diagnostic and therapeutic relevance.
A heightened inflammatory response occurs following cardiac surgery. The perioperative use of glucocorticoids has been advocated as a method to improve postoperative outcomes. Randomized prospective studies to quantify the effect of methylprednisolone on perioperative outcomes in neonatal cardiac surgery have not been performed. We sought to determine whether pre-operative methylprednisolone would improve postoperative recovery in neonates requiring cardiac surgery.
Neonates scheduled for cardiac surgery were randomly assigned to receive either Two Dose (8 hours preoperatively and operatively; n=39) or Single Dose (operatively; n=37) methylprednisolone (30 mg/kg/dose) in a double-blind, placebo-controlled trial. The primary outcome was the incidence of low cardiac output syndrome (standardized score) or death 36 hours postoperatively. Secondary outcomes were death at 30 days, interlukin-6 levels, inotropic score, fluid balance, serum creatinine, and ICU and hospital stay.
Preoperative plasma levels of the inflammatory cytokine interlukin-6 were reduced by 2-fold (p<0.001) in the Two Dose methylprednisolone group, consistent with the anti-inflammatory effects of methylprednisolone. However, the incidence of low cardiac output syndrome was 46% (17/37) in the Single Dose and 38% (15/39) in the Two Dose methylprednisolone groups (p=0.51). Two Dose methylprednisolone was associated with a higher serum creatinine (0.61±0.18 vs. 0.53±0.12 mg/dL, p=0.03), and poorer postoperative diuresis (−96±49 mL, p=0.05). Inotropic requirement, duration of mechanical ventilation, ICU, and hospital stay did not differ between the 2 groups.
Combined preoperative and intraoperative use of glucocorticoids in neonatal cardiac surgery does not favorably affect early clinical outcomes, and may exacerbate perioperative renal dysfunction.
Increasing evidence points to a direct role for altered microRNA (miRNA or miR) expression levels in cardiovascular remodeling and disease progression. While alterations in miR expression levels have been directly linked to cardiac hypertrophy, fibrosis, and remodeling, their role in regulating gene expression during thoracic aortic aneurysm (TAA) development has yet to be explored.
Methods and Results
The present study examined miR expression levels in aortic tissue specimens collected from patients with ascending TAAs by quantitative real-time PCR, and observed decreased miR expression (miRs -1, -21, -29a, -133a, and -486) as compared to normal aortic specimens. A significant relationship between miR expression levels (miRs -1, -21, -29a, and -133a) and aortic diameter was identified; as aortic diameter increased, miR expression decreased. Using a bioinformatics approach, members of the matrix metalloproteinase (MMP) family, proteins involved in TAA development, were examined for putative miR binding sites. MMP-2 and MMP-9 were identified as potential targets for miR-29a and miR-133a respectively, and MMP-2 was subsequently verified as a miR-29a target in vitro. A significant inverse relationship between miR-29a and total MMP-2 was then identified in the clinical TAA specimens.
These findings demonstrate altered miR expression patterns in clinical TAA specimens, suggesting that the loss of specific miR expression may allow for the elaboration of specific MMPs capable of driving aortic remodeling during TAA development. Importantly, these data suggest that these miRs have biological and clinical relevance to the behavior of TAAs, and may provide significant targets for therapeutic and diagnostic applications.
aneurysm; thoracic aorta; microRNA; MMP; remodeling
The present study examined a cardiac passive restraint device which applies epicardial pressure (HeartNetTM Implant) in a clinically relevant model of dilated cardiomyopathy (DCM) to determine effects on hemodynamic and myocardial blood flow patterns.
DCM was established in 10 pigs (3 weeks atrial pacing, 240 beats per minute). Hemodynamic parameters and regional left ventricle (LV) blood flow were measured under baseline conditions and following acute HeartNet (Paracor Medical Inc, Sunnyvale, CA) placement. Measurements were repeated following adenosine infusion, allowing maximal coronary vasodilation and coronary flow reserve determination.
LV dilation and systolic dysfunction occurred relative to baseline as measured by echocardiography. LV end diastolic dimension increased and LV fractional shortening decreased (3.8±0.1 vs 6.1±0.2cm and 31.6±0.5 vs 16.2±2.1%, both p<0.05 respectively) consistent with the DCM phenotype. The HeartNet was successfully deployed without arrhythmias and a computed median mid-LV epicardial pressure of 1.4 mmHg was applied by the HeartNet throughout the cardiac cycle. Acute HeartNet placement did not adversely affect steady state hemodynamics. With the HeartNet in place, coronary reserve was significantly blunted.
In a large animal model of DCM, the cardiac passive restraint device did not appear to adversely affect basal resting myocardial blood flow. However, following acute HeartNet placement, LV maximal coronary reserve was blunted. These unique results suggest that cardiac passive restraint devices which apply epicardial transmural pressure can alter myocardial blood flow patterns in a DCM model. Whether this blunting of coronary reserve holds clinical relevance with chronic passive restraint device placement remains unestablished.
While localized delivery of biocomposite materials, such as calcium hydroxyapatite (CHAM), have been demonstrated to potentially attenuate adverse LV remodeling post-myocardial infarction (MI), the underlying biological mechanisms for this effect remain unclear. This study tested the hypothesis that targeted CHAM injections would alter proteolytic pathways (matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs)), and be associated with parameters of post-MI LV remodeling.
Methods and Results
MI was induced in adult sheep followed by 20 targeted injections of a total volume of 1.3 mL (n=6) or 2.6 mL of CHAM (n=5), or saline (n=13), and LV end-diastolic volume (EDV) and MMP/TIMP profiles in the MI region were measured at 8 weeks post-MI. LV EDV decreased with 2.6 mL CHAM vs MI Only (105.4±7.5 vs 80.6±4.2 respectively, p<0.05) but not with 1.3 mL CHAM (94.5±5.0, p=0.32). However, MI thickness increased by 2-fold in both CHAM groups compared to MI Only (p<0.05). MMP-13 increased 40-fold in the MI Only group (p<0.05) but fell by over 6-fold in both CHAM groups (p<0.05). MMP-7 increased approximately 1.5-fold in the MI Only group (p<0.05) but decreased to referent control values in both CHAM groups in the MI region (p<0.05). Collagen content was reduced by approximately 30% in the CHAM groups compared to MI Only (p<0.05).
Differential effects on LV remodeling and MMP/TIMP profiles occurred with CHAM. Thus, targeted injections of a biocomposite material can favorably affect the post-MI remodeling process and therefore holds promise as a treatment strategy in and of itself, or as a matrix with potentially synergistic effects with localized pharmacologic or cellular therapies.
Infarct Expansion; Matrix Metalloproteinase; Tissue Inhibitor of Matrix Metalloproteinases
Matrix metalloproteinases (MMPs) are known to modulate left ventricular (LV) remodeling after a myocardial infarction (MI). However, the temporal and spatial variation of MMP activation and their relationship to mechanical dysfunction post MI remains undefined.
Methods and Results
MI was surgically induced in pigs (n=23) and cine MR and dual isotope hybrid SPECT/CT imaging obtained using thallium-201 (201Tl) and a technetium-99m labeled MMP targeted tracer (99mTc-RP805) at 1, 2 and 4 weeks post MI along with controls (n=5). Regional myocardial strain was computed from MR images and related to MMP zymography and ex vivo myocardial 99mTc-RP805 retention. MMP activation as assessed by in vivo and ex vivo 99mTc-RP805 imaging/retention studies was increased nearly 5-fold within the infarct region at 1 week post-MI and remained elevated up to 1 month post-MI. The post-MI change in LV end-diastolic volumes was correlated with MMP activity (y=31.34e0.48x, p=0.04). MMP activity was increased within the border and remote regions early post-MI, but declined over 1 month. There was a high concordance between regional 99mTc-RP805 uptake and ex vivo MMP-2 activity.
A novel, multimodality non-invasive hybrid SPECT/CT imaging approach was validated and applied for in vivo evaluation of MMP activation in combination with cine MR analysis of LV deformation. Increased 99mTc-RP805 retention was seen throughout the heart early post-MI and was not purely a reciprocal of 201Tl perfusion. 99mTc-RP805 SPECT/CT imaging may provide unique information regarding regional myocardial MMP activation and predict late post-MI LV remodeling.
MMP; LV remodeling; spatiotemporal imaging