Myocardial Matrix Metalloproteinases Research Articles

Dual Angiotensin Receptor-Neprilysin Inhibition With Sacubitril/Valsartan Attenuates Systolic Dysfunction in Experimental Doxorubicin-Induced Cardiotoxicity

BackgroundDoxorubicin (DOX) induces cardiotoxicity in part by activation of matrix metalloproteinases (MMPs). Sacubitril/valsartan (Sac/Val) exerts additive cardioprotective actions over renin-angiotensin-aldosterone inhibitors in preclinical models of myocardial infarction and in heart failure patients. We hypothesized that Sac/Val would be more cardioprotective than Val in a rodent model of progressive DOX-induced cardiotoxicity, and this benefit would be associated with modulation of MMP activation. ObjectivesWe sought to investigate the efficacy of Sac/Val for the treatment of anthracycline-induced cardiotoxicity. MethodsMale Wistar rats received DOX intraperitoneally (15 mg/kg cumulative) or saline over 3 weeks. Following the first treatment, control animals were gavaged daily with water (n = 25), while DOX-treated animals were gavaged daily with water (n = 25), Val (31 mg/kg; n = 25) or Sac/Val (68 mg/kg; n = 25) for either 4 or 6 weeks. Echocardiography was performed at baseline, and 4 and 6 weeks after DOX initiation. In addition, myocardial MMP activity was assessed with 99mTc-RP805, and cardiotoxicity severity was assessed by histology at these time points in a subgroup of animals. ResultsLeft ventricular ejection fraction decreased by 10% at 6 weeks in DOX and DOX + Val rats (both p < 0.05), while this reduction was attenuated in DOX + Sac/Val rats. MMP activity was increased at 6 weeks by 76% in DOX-alone rats, and tended to increase in DOX + Val rats (36%; p = 0.051) but was similar in DOX + Sac/Val rats as compared with time-matched control animals. Both therapies attenuated histological evidence of cellular toxicity and fibrosis (p < 0.05). ConclusionsSac/Val offers greater protection against left ventricular remodeling and dysfunction compared with standard angiotensin receptor blocker therapy in a rodent model of progressive DOX-induced cardiotoxicity.

Matrix Metalloproteinases Increase Because of Hypoperfusion in Obstructive Hypertrophic Cardiomyopathy

Myocardial fibrosis (MF) is considered a result of microvascular dysfunction in patients with hypertrophic cardiomyopathy. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), capable of degrading collagen, directly participate in the development of MF. First we investigated the relationships among MF, microvascular rarefaction, and MMPs. Then we assessed the prognostic value of MF-related circulating biomarkers. Fifty-five obstructive hypertrophic cardiomyopathy (HOCM) patients were enrolled after surgical myectomy. Myocardial samples were performed with Masson's trichrome staining and immunohistochemical procedures for collagen volume fraction and microvascular density, respectively. Enzyme-linked immunosorbent assays were used to assess myocardial and plasma of MMP-2, MMP-9, and TIMP-1 and plasma C-terminal propeptide of procollagen type Ⅰ (PICP) and C-terminal telopeptide of type Ⅰ collagen (ICTP) levels. The composite cardiovascular endpoint consisted of new-onset atrial fibrillation, heart failure requiring hospitalization, and all-cause death. In HOCM patients microvascular density was associated with the myocardial MMP-2/TIMP-1 ratio (r= -0.348, P= .009), whereas no correlation was found between collagen volume fraction and myocardial MMPs. During the 44-month follow-up 6 patients experienced a cardiovascular endpoint. The plasma PICP/ICTP ratio and MMP-2/TIMP-1 ratio were the 2 strongest prognostic makers. In multivariable analyses high PICP/ICTP and MMP-2/TIMP-1 ratios remained independent predictors of cardiovascular outcomes after adjusting for clinical confounders (hazard ratios, 12.683 [P= .021] and 17.037 [P= .027], respectively). In HOCM patients the myocardial MMP-2/TIMP-1 ratio was elevated because of microvascular rarefaction but may not be responsible for MF. High plasma PICP/ICTP and MMP-2/TIMP-1 ratios are independent predictors of adverse outcomes in HOCM patients.

MMP inhibitors attenuate doxorubicin cardiotoxicity by preventing intracellular and extracellular matrix remodelling.

Heart failure is a major complication in cancer treatment due to the cardiotoxic effects of anticancer drugs, especially from the anthracyclines such as doxorubicin (DXR). DXR enhances oxidative stress and stimulates matrix metalloproteinase-2 (MMP-2) in cardiomyocytes. We investigated whether MMP inhibitors protect against DXR cardiotoxicity given the role of MMP-2 in proteolyzing sarcomeric proteins in the heart and remodelling the extracellular matrix. Eight-week-old male C57BL/6J mice were treated with DXR weekly with or without MMP inhibitors doxycycline or ONO-4817 by daily oral gavage for 4 weeks. Echocardiography was used to determine cardiac function and left ventricular remodelling before and after treatment. MMP inhibitors ameliorated DXR-induced systolic and diastolic dysfunction by reducing the loss in left ventricular ejection fraction, fractional shortening, and E'/A'. MMP inhibitors attenuated adverse left ventricular remodelling, reduced cardiomyocyte dropout, and prevented myocardial fibrosis. DXR increased myocardial MMP-2 activity in part also by upregulating N-terminal truncated MMP-2. Immunogold transmission electron microscopy showed that DXR elevated MMP-2 levels within the sarcomere and mitochondria which were associated with myofilament lysis, mitochondrial degeneration, and T-tubule distention. DXR-induced myofilament lysis was associated with increased titin proteolysis in the heart which was prevented by ONO-4817. DXR also increased the level and activity of MMP-2 in human embryonic stem cell-derived cardiomyocytes, which was reduced by ONO-4817. MMP-2 activation is an early event in DXR cardiotoxicity and contributes to myofilament lysis by proteolyzing cardiac titin. Two orally available MMP inhibitors ameliorated DXR cardiotoxicity by attenuating intracellular and extracellular matrix remodelling, suggesting their use may be a potential prophylactic strategy to prevent heart injury during chemotherapy.

Application of Hybrid Matrix Metalloproteinase-Targeted and Dynamic 201Tl Single-Photon Emission Computed Tomography/Computed Tomography Imaging for Evaluation of Early Post-Myocardial Infarction Remodeling.

The induction of matrix metalloproteinases (MMPs) and reduction in tissue inhibitors of MMPs (TIMPs) plays a role in ischemia/reperfusion (I/R) injury post-myocardial infarction (MI) and subsequent left ventricular remodeling. We developed a hybrid dual isotope single-photon emission computed tomography/computed tomography approach for noninvasive evaluation of regional myocardial MMP activation with 99mTc-RP805 and dynamic 201Tl for determination of myocardial blood flow, to quantify the effects of intracoronary delivery of recombinant TIMP-3 (rTIMP-3) on I/R injury. Studies were performed in control pigs (n=5) and pigs following 90-minute balloon occlusion-induced ischemia/reperfusion (I/R) of left anterior descending artery (n=9). Before reperfusion, pigs with I/R were randomly assigned to intracoronary infusion of rTIMP-3 (1.0 mg/kg; n=5) or saline (n=4). Three days post-I/R, dual isotope imaging was performed with 99mTc-RP805 and 201Tl along with contrast cineCT to assess left ventricular function. The ischemic to nonischemic ratio of 99mTc-RP805 was significantly increased following I/R in saline group (4.03±1.40), and this ratio was significantly reduced with rTIMP-3 treatment (2.22±0.57; P=0.03). This reduction in MMP activity in the MI-rTIMP-3 treatment group was associated with an improvement in relative MI region myocardial blood flow compared with the MI-saline group and improved myocardial strain in the MI region. We have established a novel hybrid single-photon emission computed tomography/computed tomography imaging approach for the quantitative assessment of regional MMP activation, myocardial blood flow, and cardiac function post-I/R that can be used to evaluate therapeutic interventions such as intracoronary delivery of rTIMP-3 for reduction of I/R injury in the early phases of post-MI remodeling.

Low-intensity aerobic exercise improves cardiac remodelling of adult spontaneously hypertensive rats.

We evaluated the influence of aerobic training on cardiac remodeling in untreated spontaneously hypertensive rats (SHR). Four experimental groups were used: sedentary (W‐SED, n=27) and trained (WEX, n=31) normotensive Wistar rats, and sedentary (SHR‐SED, n=27) and exercised (SHR‐EX, n=32) hypertensive rats. At 13 months old, trained groups underwent treadmill exercise five days a week for four months. Statistical analysis: ANOVA or Kruskal‐Wallis. Exercised groups had higher physical capacity. Hypertensive groups presented left ventricular (LV) concentric hypertrophy with impaired function. Left atrium diameter, LV posterior wall thickness and relative thickness, and isovolumetric relaxation time were lower in SHR‐EX than SHR‐SED. Interstitial collagen fraction and Type I‐Type III collagen ratio were higher in SHR‐SED than W‐SED. In SHR‐EX these parameters had intermediate values between W‐EX and SHRSED with no differences between either group. Myocardial matrix metalloproteinase‐2 activity, evaluated by zymography, was higher in SHR‐SED than W‐SED and SHR‐EX. TIMP‐2 was higher in hypertensive than normotensive groups. In conclusion, low intensity aerobic exercise reduces left atrium dimension and LV posterior wall thickness, and improves functional capacity, diastolic function, and metalloproteinase‐2 activity in adult SHR.

Alterations in myocardial connexin-43 and matrix metalloproteinase-2 signaling in response to pregnancy and oxygen deprivation of Wistar rats: a pilot study 1.

Two important aspects of cardiac adaptive response to pregnancy have been studied in normal as well as hypoxic conditions: (1) intercellular signaling mediated by myocardial connexin-43 (Cx43) that is crucial to synchronize heart function; (2) extracellular signaling mediated by matrix metalloproteinase-2 (MMP-2) that is an early marker of extracellular matrix remodeling. Myocardial Cx43 distribution and functional capillary density were determined as well. Hypoxia was induced by exposure of rats to 10.5% O2 and 89.5% N2 in a hermetically sealed chamber. Findings showed that pregnancy resulted in a significant increase of Cx43 protein expression, its functional phosphorylated forms, and enhanced capillary density while did not affect either expression of total MMP-2 or its activity. Maternal hypoxia for 12 or 16 h did not affect elevated Cx43 but enhanced its distribution on lateral sides of the cardiomyocytes. In contrast, hypoxia of nonpregnant rats resulted in upregulation of Cx43, its lateral distribution, and enhanced capillary density. Hypoxia did not affect myocardial MMP-2 either in pregnant or nonpregnant rats. Cardiac adaptive response to pregnancy is accompanied by enhanced Cx43 without changes in MMP-2 signaling. Pregnant rat heart is tolerant to short-term hypoxemia, while nonpregnant rat heart reacts by upregulation of Cx43 and increased capillary density.

Acute Doxorubicin-Induced Cardiotoxicity is Associated with Matrix Metalloproteinase-2 Alterations in Rats

Background: Doxorubicin can cause cardiotoxicity. Matrix metalloproteinases (MMP) are responsible for degrading extracellular matrix components which play a role in ventricular dilation. Increased MMP activity occurs after chronic doxorubicin treatment. In this study we evaluated in vivo and in vitro cardiac function in rats with acute doxorubicin treatment, and examined myocardial MMP and inflammatory activation, and gene expression of proteins involved in myocyte calcium transients. Methods: Wistar rats were injected with doxorubicin (Doxo, 20 mg/kg) or saline (Control). Echocardiogram was performed 48 h after treatment. Myocardial function was assessed in vitro in Langendorff preparation. Results: In left ventricle, doxorubicin impaired fractional shortening (Control 0.59±0.07; Doxo 0.51±0.05; p<0.001), and increased isovolumetric relaxation time (Control 20.3±4.3; Doxo 24.7±4.2 ms; p=0.007) and myocardial passive stiffness. MMP-2 activity, evaluated by zymography, was increased in Doxo (Control 141338 ± 8924; Doxo 188874 ± 7652 arbitrary units; p<0.001). There were no changes in TNF-α, INF-γ, IL-10, and ICAM-1 myocardial levels. Expression of phospholamban, Serca-2a, and ryanodine receptor did not differ between groups. Conclusion: Acute doxorubicin administration induces in vivo left ventricular dysfunction and in vitro increased myocardial passive stiffness in rats. Cardiac dysfunction is related to myocardial MMP-2 activation. Increased inflammatory stimulation or changed expression of the proteins involved in intracellular calcium transients is not involved in acute cardiac dysfunction.

Loss of NOX2 (gp91phox) prevents oxidative stress and progression to advanced heart failure

Oxidative stress plays a key pathogenic role in experimental and human heart failure. However, the source of ROS (reactive oxygen species) is a key determinant of the cardiac adaptation to pathological stressors. In the present study, we have shown that human dilated cardiomyopathy is associated with increased NOX2 (NADPH oxidase 2) levels, increased oxidative stress with adverse myocardial remodelling and activation of MAPKs (mitogen-activated protein kinases). Advanced heart failure in mice was also associated with increased NOX2 levels. Furthermore, we have utilized the pressure-overload model to examine the role of NOX2in advanced heart failure. Increased cardiomyocyte hypertrophy and myocardial fibrosis in response to pressure overload correlated with increased oxidative stress, and loss of NOX2 prevented the increase in oxidative stress, development of cardiomyocyte hypertrophy, myocardial fibrosis and increased myocardial MMP (matrix metalloproteinase) activity in response to pressure overload. Consistent with these findings, expression of disease markers revealed a marked suppression of atrial natriuretic factor, β-myosin heavy chain, B-type natriuretic peptide and α-skeletal actin expression in pressure-overloaded hearts from NOX2-deficient mice. Activation of MAPK signalling, a well-known mediator of pathological remodelling, was lowered in hearts from NOX2-deficient mice in response to pressure overload. Functional assessment using transthoracic echocardiography and invasive pressure-volume loop analysis showed a marked protection in diastolic and systolic dysfunction in pressure-overloaded hearts from NOX2-deficient mice. Loss of NOX2 prevented oxidative stress in heart disease and resulted in sustained protection from the progression to advanced heart failure. Our results support a key pathogenic role of NOX2in murine and human heart failure, and specific therapy antagonizing NOX2 activity may have therapeutic effects in advanced heart failure.

Expression of the tissue inhibitor of metalloproteinase-3 by transplanted VSMCs modifies heart structure and function after myocardial infarction

ObjectivesExtracellular matrix (ECM) remodelling is a critical aspect of cardiac remodelling following myocardial infarction. Tissue inhibitors of metalloproteinases (TIMPs) are physiological inhibitors of matrix metalloproteinases (MMPs) that degrade the ECM proteins. TIMP-3 is highly expressed in the heart and is markedly downregulated in patients with ischaemic cardiomyopathy. Cell-based gene therapy can enhance the effects of cell transplantation by temporally and spatially regulating the release of the gene product. The purpose of this study was to investigate the role of TIMP-3 gene-transfected vascular smooth muscle cells (VSMCs) in modifying heart structure and function in rats when transplanted 3days after myocardial infarction (MI). MethodsAnesthetised rats were subjected to coronary artery ligation followed 3days later by thoracotomy and transplantation of TIMP-3 gene-transfected VSMCs, untransfected VSMCs or medium injected directly into the ischaemic myocardium. We assessed left ventricular structure and function by echocardiography and morphometry, and measured the levels of myocardial matrix metalloproteinase-2 and -9 (MMP-2, MMP-9), TIMP-3 and tumour necrosis factor-α (TNF-α) at 4weeks post-myocardial infarction. ResultsTransplantation of TIMP-3 gene-transfected VSMCs and untransfected VSMCs significantly decreased scar expansion and ventricular dilatation 25days post-transplantation (4weeks after MI). MMPs and TNF-α levels were reduced in the transplantation groups when compared to the group that was given an injection of medium only. Transplantation of TIMP-3 gene-transfected VSMCs was more effective in preventing progressive cardiac dysfunction, ventricular dilatation and in reducing MMP-2, MMP-9 and TNF-α levels when compared to the transplantation of untransfected VSMCs. ConclusionsTIMP-3 gene transfection was associated with attenuated left ventricular dilation and recovery of systolic function after MI compared with the control. TIMP-3 transfection enhanced the effects of transplanted VSMCs in rats by inhibiting matrix degradation and inflammatory cytokine expression, leading to improved myocardial remodelling.

Postresuscitation Administration of Doxycycline Preserves Cardiac Contractile Function in Hypoxia-Reoxygenation Injury of Newborn Piglets*

Cardiac injury is common in asphyxiated neonates and is associated with matrix metalloproteinase-2 activation. Although studies have demonstrated the cardioprotective effects of matrix metalloproteinase inhibition, this has not been tested in clinically translatable models of hypoxia-reoxygenation injury. We aimed to elucidate the effect of doxycycline, a matrix metalloproteinase inhibitor, on cardiac injury and functional recovery in a swine model of neonatal hypoxia-reoxygenation. Thirty-three newborn piglets were acutely instrumented for continuous monitoring of cardiac output and systemic arterial pressure. After stabilization, normocapnic alveolar hypoxia (10-15% oxygen) was instituted for 2 hours followed by 4 hours of normoxic reoxygenation. Piglets were blindly, block randomized to receive IV boluses of normal saline (control) and doxycycline at 5 minutes of reoxygenation (n = 7/group). Sham-operated piglets (n = 5) received no hypoxia-reoxygenation. Markers of myocardial injury (plasma and myocardial tissue troponin I; myocardial lactate) and oxidative stress (lipid hydroperoxides) were measured by enzyme-linked immunosorbent assay and Western blot. Myocardial matrix metalloproteinase-2 activity was quantified by gelatin zymography and immunoprecipitation. University animal laboratory. Piglets (1-4 d old, weighing 1.4-2.5 kg). IV doxycycline (3, 10, or 30 mg/kg) given during resuscitation. Hypoxic piglets had cardiogenic shock (cardiac output 58% ± 1% of baseline), hypotension (systemic arterial pressure 31 ± 1 mm Hg), and acidosis (pH 7.02 ± 0.02). Doxycycline improved cardiac and stroke volume index with no chronotropic effect in doxycycline-treated piglets compared with controls. Systemic arterial pressure was higher and the pulmonary artery pressure/systemic arterial pressure ratio was lower in doxycycline groups, with reduced levels of markers of myocardial injury and oxidative stress in doxycycline-treated piglets compared with controls. Negative correlations were found between markers of myocardial injury (plasma troponin I, myocardial lactate) and functional recovery and between myocardial tissue and plasma troponin I. Doxycycline-treated piglets had lower myocardial matrix metalloproteinase-2 activity compared with controls. Postresuscitation administration of doxycycline attenuates cardiac injury and improves functional recovery in newborn piglets with hypoxia-reoxygenation.

Secreted Frizzled-related protein 2 as a target in antifibrotic therapeutic intervention

Progressive fibrosis is a pathological hallmark of many chronic diseases responsible for organ failure. Although there is currently no therapy on the market that specifically targets fibrosis, the dynamic fibrogenic process is known to be regulated by multiple soluble mediators that may be therapeutically intervened. The failing hamster heart exhibits marked fibrosis and increased expression of secreted Frizzled-related protein 2 (sFRP2) amenable to reversal by mesenchymal stem cell (MSC) therapy. Given the previous demonstration that sFRP2-null mice subjected to myocardial infarction exhibited reduced fibrosis and improved function, we tested whether antibody-based sFRP2 blockade might counteract the fibrogenic pathway and repair cardiac injury. Cardiomyopathic hamsters were injected intraperitoneally twice a week each with 20 μg of sFRP2 antibody. Echocardiography, histology, and biochemical analyses were performed after 1 mo. sFRP2 antibody increased left ventricular ejection fraction from 40 ± 1.2 to 49 ± 6.5%, whereas saline and IgG control exhibited a further decline to 37 ± 0.9 and 31 ± 3.2%, respectively. Functional improvement is associated with a ∼50% reduction in myocardial fibrosis, ∼65% decrease in apoptosis, and ∼75% increase in wall thickness. Consistent with attenuated fibrosis, both MSC therapy and sFRP2 antibody administration significantly increased the activity of myocardial matrix metalloproteinase-2. Gene expression analysis of the hamster heart and cultured fibroblasts identified Axin2 as a downstream target, the expression of which was activated by sFRP2 but inhibited by therapeutic intervention. sFRP2 blockade also increased myocardial levels of VEGF and hepatocyte growth factor (HGF) along with increased angiogenesis. These findings highlight the pathogenic effect of dysregulated sFRP2, which may be specifically targeted for antifibrotic therapy.

Moderate inhibition of myocardial matrix metalloproteinase-2 by ilomastat is cardioprotective

Pharmacological inhibition of matrix metalloproteinase-2 (MMP-2) is a promising target for acute cardioprotection against ischemia/reperfusion injury. Therefore, here we investigated if the MMP inhibitor ilomastat administered either before ischemia or before reperfusion is able to reduce infarct size via inhibition of MMP-2, the most abundant MMP in the rat heart.Infarct-size limiting effect of ilomastat (0.3–6.0μmol/kg) was tested in an in vivo rat model of myocardial infarction induced by 30min coronary occlusion/120min reperfusion. Ilomastat at 0.75 and 1.5μmol/kg decreased infarct size significantly as compared to the vehicle-treated (dimethyl sulfoxide) group (from 66.1±4.6% to 45.3±7.0% and 46.7±5.5% of area at risk, p<0.0.5, respectively), when administered 5min before the onset of ischemia. Ilomastat at 6.0μmol/kg significantly reduced infarct size from its control value of 65.4±2.5% to 52.8±3.7% of area at risk (p<0.05), when administered 5min before the onset of reperfusion. Area at risk was not significantly affected by ilomastat treatments. To further assess the cytoprotective effect of ilomastat, primary cardiomyocytes isolated from neonatal rats were subjected to 240min simulated ischemia followed by 120min simulated reperfusion in the presence of ilomastat (5nM–5μM). Ilomastat at 500nM and 5μM significantly increased cell viability when compared to vehicle treated group. To assess the in situ MMP-2 inhibitory effect of ilomastat, in separate experiments in situ zymography was performed in cardiomyocytes. The cytoprotective concentration of ilomastat (500nM) showed a moderate (approximately 25%) inhibition of intracellular MMP-2 in ischemic/reperfused cardiomyocytes. In these cells, MMP-2 immunostaining showed a 90% colocalization with the in situ gelatinolytic activity.We conclude that the MMP inhibitor ilomastat reduces infarct size when administered either before the onset of ischemia or before the onset of reperfusion in vivo. Furthermore, this is the first demonstration that a moderate inhibition of intracellular MMP-2 is sufficient to confer cardiocytoprotection.

Doxycycline Prevents Acute Pulmonary Embolism-Induced Mortality and Right Ventricular Deformation in Rats

Acute pulmonary embolism (APE) is a critical cardiopulmonary condition associated with right ventricular (RV) failure and death. While pharmacological inhibition of matrix metalloproteinases (MMPs) attenuated APE-induced hemodynamic alterations, no previous study has evaluated whether this approach decreases APE-induced mortality and RV deformation. We tested this hypothesis in rats. Wistar rats received an intraperitoneal injection of 30 mg/kg doxycycline (or saline) and after 30 min a sterile suspension of 300 μm microsphere (21 mg/kg or saline) was injected into the tail vein. After 24 h, surviving animals were killed and the RVs were collected and used for histological and morphometric analyses. RV samples were also homogenized and assayed by SDS-polyacrilamide gel electrophoresis gelatin zymography to evaluate MMP-2 and MMP-9 activity. In situ zymography was carried out in RV to assess MMP activity and neutrophil accumulation in myocardial tissue was determined by myeloperoxidase activity measurement. Dihydroethidium was used to assess RV reactive oxygen species concentrations. APE caused 72.5% mortality during the first hour of follow up. Pretreatment with doxycycline was associated with significant decrease in APE-induced mortality rate to 50% (P<0.05). Embolized animals showed significant RV dilation, and pretreatment with doxycycline blunted this alteration (P<0.05). APE increased the number of RV neutrophils and MMP-9 levels (P<0.05). Pretreatment with doxycycline blunted APE-induced increases in RV myocardial ROS concentrations and MMP gelatinolytic activity (both P<0.05). These findings show that MMP inhibition with doxycycline protects against APE-induced mortality and RV enlargement. These beneficial effects are probably due to attenuation of APE-induced oxidative stress and increases in ventricular proteolytic activity and suggest that doxycycline may have promising protective effects in patients with APE.

Doxycycline Attenuates Cardiac Injury and Improves Cardiac Function with Inhibition of Myocardial Matrix Metalloproteinase (MMP)‐2 in a Swine Model of Hypoxia‐ Reoxygenation (H‐R)

BackgroundCardiac dysfunction is common in asphyxiated neonates. Although small animal studies support attenuation of cardiac injury with the known MMP inhibitor doxycycline in H‐R, this effect has not been tested in a clinically relevant large animal model of H‐R. We hypothesized that doxycycline would improve the recovery of cardiac function in newborn piglets with H‐R.MethodsPiglets were instrumented for hemodynamic monitoring and subjected to 2 hr of hypoxia followed by 4 hr of normoxic reoxygenation. Piglets were blindly randomized to receive i.v. saline or doxycycline (3, 10, or 30 mg/kg) 5 min into reoxygenation (n=7/group). Sham piglets (n=5) received no H‐R. Markers of myocardial injury (serum and myocardial tissue troponin; myocardial lactate) and oxidative stress (lipid hydroperoxides) were measured by ELISA and Western blot. Myocardial MMP‐2 activity was quantified by gelatin zymography.ResultsDoxycycline dose‐dependently improved cardiac (CI) and stroke volume (SVI) index (30mg/kg: 84±3 (SEM)% and 78±5% of baseline vs. 65±3% and 50±5% in controls [p&lt;0.05]). Markers of myocardial injury, oxidative stress and MMP‐2 activity were improved in doxycycline groups vs. control [all p&lt;0.05]. Significant correlations were found between markers of myocardial injury and CI and SVI recovery (r =−0.5, p&lt;0.01), with a negative correlation also observed between myocardial tissue and serum troponins (r =−0.4, p=0.02).ConclusionsDoxycycline attenuates cardiac injury and improves functional recovery in newborn pigs with H‐R.Funded by: Canadian Institutes of Health Research and the Women and Children's Health Research Institute

Expression of matrix metalloproteinases, their inhibitors, and lysyl oxidase in myocardial samples from dogs with end-stage systemic and cardiac diseases

To compare the degree of mRNA expression for matrix metalloproteinases (MMPs), tissue inhibitors (TIMPs), and lysyl oxidase in myocardial samples from dogs with cardiac and systemic diseases and from healthy control dogs. Myocardial samples from the atria, ventricles, and septum of 8 control dogs, 6 dogs with systemic diseases, 4 dogs with dilated cardiomyopathy (DCM), and 5 dogs with other cardiac diseases. Degrees of mRNA expression for MMP-1, -2, -3, -9, and -13; TIMP-1, -2, -3, and -4; and lysyl oxidase were measured via quantitative real-time PCR assay. Histologic examination of the hearts was performed to identify pathological changes. In myocardial samples from control dogs, only TIMP-3 and TIMP-4 mRNA expression was detected, with a significantly higher degree in male versus female dogs. In dogs with systemic and cardiac diseases, all investigated markers were expressed, with a significantly higher degree of mRNA expression than in control dogs. Furthermore, the degree of expression for MMP-2, TIMP-1, and TIMP-2 was significantly higher in dogs with DCM than in dogs with systemic diseases and cardiac diseases other than DCM. Expression was generally greater in atrial than in ventricular tissue for MMP-2, MMP-13, and lysyl oxidase in samples from dogs with atrial fibrillation. Degrees of myocardial MMP, TIMP, and lysyl oxidase mRNA expression were higher in dogs with cardiac and systemic diseases than in healthy dogs, suggesting that expression of these markers is a nonspecific consequence of end-stage diseases. Selective differences in the expression of some markers may reflect specific pathogenic mechanisms and may play a role in disease progression, morbidity and mortality rates, and treatment response.

Early activation of myocardial matrix metalloproteinases and degradation of cardiac troponin I after experimental subarachnoid hemorrhage

BackgroundMatrix metalloproteinases (MMPs) are involved in acute myocardial dysfunction by degrading several intracellular contractile proteins, including cardiac troponin I (cTnI). Here, we examined the temporal profiles of MMPs and cTnI in plasma and myocardial tissue in the acute stage of subarachnoid hemorrhage (SAH). Materials and methodsSAH was induced by the endovascular suture method in rats. Intracranial pressure and left ventricular (LV) function were recorded. Plasma cTnI and MMPs were measured at 0, 5, 15, 30, 60, 120, and 180 minutes after SAH. Myocardial cTnI and MMP activities were quantified at 30, 60 and 180 min after SAH from homogenized hearts. ResultsSAH-induced rats showed a marked decline in -LV dP/dtmax (index of LV diastolic function). Plasma samples revealed a noticeable increase in cTnI and pro-MMP-9 activities over the course of 180 minutes. In myocardial tissue, there was a marked increase in pro-MMP-9, pro-MMP-2 activities and expression of activated MMP-2. Western blot analysis revealed a striking decrease in cTnI content and increase in cTnI degradation in myocardium. Simultaneous cTnI depletion and MMP-2 expression in myocardium was detected by immunohistochemistry as early as 30 minutes after SAH. MMPs correlated with -LV dP/dtmax (% of baseline) both in plasma and in myocardial tissue. Furthermore, activated MMP-2 activity correlated positively with cTnI degradation in myocardium. ConclusionsEarly activation of MMPs was observed in myocardium and plasma following SAH. Activated MMP-2 may regulate proteolytic cTnI and contribute to myocardium stunning injury in SAH rats.

Regulation of myocardial matrix metalloproteinase expression and activity by cardiac fibroblasts

AbstractCardiac fibroblasts (CF) play a key role in orchestrating the structural remodeling of the myocardium in response to injury or stress, in part through direct regulation of extracellular matrix (ECM) turnover. The matrix metalloproteinases (MMPs) are a family of over 25 zinc‐dependent proteases that together have the capacity to degrade all the protein components of the ECM. Fibroblasts are a major source of several MMPs in the heart, thereby representing a viable therapeutic target for regulating ECM turnover in cardiac pathologies characterized by adverse remodeling, such as myocardial infarction, cardiomyopathy, hypertension and heart failure. This review summarizes current knowledge on the identity and regulation of MMPs expressed by CF and discusses future directions for reducing adverse myocardial remodeling by modulating the expression and/or activity of CF‐derived MMPs. © 2012 IUBMB IUBMB Life, 2012.

Regulation of myocardial matrix metalloproteinase expression and activity by cardiac fibroblasts

Cardiac fibroblasts (CF) play a key role in orchestrating the structural remodeling of the myocardium in response to injury or stress, in part through direct regulation of extracellular matrix (ECM) turnover. The matrix metalloproteinases (MMPs) are a family of over 25 zinc-dependent proteases that together have the capacity to degrade all the protein components of the ECM. Fibroblasts are a major source of several MMPs in the heart, thereby representing a viable therapeutic target for regulating ECM turnover in cardiac pathologies characterized by adverse remodeling, such as myocardial infarction, cardiomyopathy, hypertension and heart failure. This review summarizes current knowledge on the identity and regulation of MMPs expressed by CF and discusses future directions for reducing adverse myocardial remodeling by modulating the expression and/or activity of CF-derived MMPs.

Regulatory T cells ameliorate cardiac remodeling after myocardial infarction

Persistent inflammatory responses participate in the pathogenesis of adverse ventricular remodeling after myocardial infarction (MI). We hypothesized that regulatory T (Treg) cells modulate inflammatory responses, attenuate ventricular remodeling and subsequently improve cardiac function after MI. Acute MI was induced by ligation of the left anterior descending coronary artery in rats. Infiltration of Foxp3(+) Treg cells was detected in the infarcted heart. Expansion of Treg cells in vivo by means of adoptive transfer as well as a CD28 superagonistic antibody (JJ316) resulted in an increased number of Foxp3(+) Treg cells in the infarcted heart. Subsequently, rats with MI showed improved cardiac function following Treg cells transfer or JJ316 injection. Interstitial fibrosis, myocardial matrix metalloproteinase-2 activity and cardiac apoptosis were attenuated in the rats that received Treg cells transfer. Infiltration of neutrophils, macrophages and lymphocytes as well as expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β were also significantly decreased, and the CD8(+) cardiac-specific cytotoxic T lymphocyte response was inhibited. Expression of interleukin (IL)-10 in the heart, however, was increased. Additional studies in vitro indicated that Treg cells directly protect neonatal rat cardiomyocytes against LPS-induced apoptosis, and this protection depends on the cell-cell contact and IL-10 expression. Furthermore, Treg cells inhibited proinflammatory cytokines production by cardiomyocytes. These data demonstrate that Treg cells serve to protect against adverse ventricular remodeling and contribute to improve cardiac function after myocardial infarction via inhibition of inflammation and direct protection of cardiomyocytes.

Endogenous Thrombospondin 1 Protects the Pressure-Overloaded Myocardium by Modulating Fibroblast Phenotype and Matrix Metabolism

The matricellular protein thrombospondin (TSP) 1 is induced after tissue injury and may regulate reparative responses by activating transforming growth factor-β, by suppressing angiogenesis and by modulating inflammation and matrix metabolism. We hypothesized that endogenous TSP-1 may be involved in the pathogenesis of cardiac remodeling in the pressure-overloaded heart. Myocardial TSP-1 expression was increased in a mouse model of pressure overload because of transverse aortic constriction. TSP-1(-/-) mice exhibited increased early hypertrophy and enhanced late dilation in response to pressure overload. Pressure-overloaded TSP-1 null mice had intense degenerative cardiomyocyte changes, exhibiting more extensive sarcomeric loss and sarcolemmal disruption when compared with wild-type hearts. Accentuated hypertrophy and cardiomyocyte injury in TSP-1(-/-) hearts was accompanied by increased myofibroblast density. However, despite a 2-fold higher infiltration of the cardiac interstitium with myofibroblasts, pressure-overloaded TSP-1 null hearts did not exhibit significantly increased collagen content when compared with wild-type hearts. The disproportionately low collagen content in TSP-1 null hearts was attributed to infiltration with abundant, but functionally defective, fibroblasts that exhibited impaired myofibroblast differentiation and reduced collagen expression in comparison with wild-type fibroblasts. Impaired myofibroblast activation in TSP-1 null hearts was associated with reduced Smad2 phosphorylation reflecting defective transforming growth factor-β signaling. Moreover, TSP-1 null hearts had increased myocardial matrix metalloproteinase 3 expression and enhanced matrix metalloproteinase 9 activation after pressure overload. TSP-1 upregulation in the pressure-overloaded heart critically regulates fibroblast phenotype and matrix remodeling by activating transforming growth factor-β signaling and by promoting matrix preservation, thus preventing chamber dilation.