Tissue inhibitor of metalloproteinase-4 deletion in mice impacts maternal cardiac function during pregnancy and postpartum.

Reversible physiological cardiac hypertrophy of the maternal heart occurs during pregnancy and involves extracellular matrix (ECM) remodeling. Previous mouse studies revealed that changes in ECM molecules accompany functional changes in the left ventricle (LV) during late pregnancy and postpartum. We evaluated the effect of global Timp4 deletion in female mice on LV functional parameters and ECM molecules during pregnancy and the postpartum period. Heart weights normalized to tibia lengths were increased in Timp4 knockout (Timp4 KO) virgin, pregnant, and postpartum day 2 mice compared with wild types. Serial echocardiography performed on pregnancy days 10, 12, and 18 and postpartum days (ppds) 2, 7, 14, 21, and 28 revealed that both wild-type and Timp4 KO mice increased end systolic and end diastolic volumes (ESV, EDV) by mid to late pregnancy compared with virgins, with EDV changes persisting through the postpartum period. When compared with wild types, Timp4 KO mice exhibited higher ejection fractions in virgins, at pregnancy days 10 and 18 and ppd2 and ppd14. High-molecular weight forms of COL1A1 and COL3A1 proteins in LV were greater in Timp4 KO virgins, and COL1A1 was higher in late pregnancy and on ppd2 compared with wild types. With exceptions, Timp4 KO mice during late pregnancy and the early postpartum period were able to maintain stroke volume similar to wild-type mice through increased ejection fraction. Although TIMP4 deletion in females exhibited altered ECM molecules, it did not adversely affect cardiac function during first pregnancies and lactation.NEW & NOTEWORTHY Pregnancy and lactation increase volume load on the heart. Defects in cardiac remodeling during pregnancy and postpartum can result in peripartum cardiomyopathy. TIMPs participate in cardiac remodeling. The present study reports the cardiac function in Timp4 knockout adult female mice during pregnancy and lactation. Timp4 knockout females at many time points have higher ejection fraction to maintain stroke volume. Global deletion of Timp4 was not detrimental to maternal heart function during first pregnancies and lactation.

Ensemble machine learning model identifies patients with HFpEF from matrix-related plasma biomarkers.

Arterial hypertension can lead to structural changes within the heart including left ventricular hypertrophy (LVH) and eventually heart failure with preserved ejection fraction (HFpEF). The initial diagnosis of HFpEF is costly and generally based on later stage remodeling; thus, improved predictive diagnostic tools offer potential clinical benefit. Recent work has shown predictive value of multibiomarker plasma panels for the classification of patients with LVH and HFpEF. We hypothesized that machine learning algorithms could substantially improve the predictive value of circulating plasma biomarkers by leveraging more sophisticated statistical approaches. In this work, we developed an ensemble classification algorithm for the diagnosis of HFpEF within a population of 480 individuals including patients with HFpEF, patients with LVH, and referent control patients. Algorithms showed strong diagnostic performance with receiver-operating-characteristic curve (ROC) areas of 0.92 for identifying patients with LVH and 0.90 for identifying patients with HFpEF using demographic information, plasma biomarkers related to extracellular matrix remodeling, and echocardiogram data. More impressively, the ensemble algorithm produced an ROC area of 0.88 for HFpEF diagnosis using only demographic and plasma panel data. Our findings demonstrate that machine learning-based classification algorithms show promise as a noninvasive diagnostic tool for HFpEF, while also suggesting priority biomarkers for future mechanistic studies to elucidate more specific regulatory roles.NEW & NOTEWORTHY Machine learning algorithms correctly classified patients with heart failure with preserved ejection fraction with over 90% area under receiver-operating-characteristic curves. Classifications using multidomain features (demographics and circulating biomarkers and echo-based ventricle metrics) proved more accurate than previous studies using single-domain features alone. Excitingly, HFpEF diagnoses were generally accurate even without echo-based measurements, demonstrating that such algorithms could provide an early screening tool using blood-based measurements before sophisticated imaging.

Interpretable machine learning predicts cardiac resynchronization therapy responses from personalized biochemical and biomechanical features.

BACKGROUND: Cardiac Resynchronization Therapy (CRT) is a widely used, device-based therapy for patients with left ventricle (LV) failure. Unfortunately, many patients do not benefit from CRT, so there is potential value in identifying this group of non-responders before CRT implementation. Past studies suggest that predicting CRT response will require diverse variables, including demographic, biomarker, and LV function data. Accordingly, the objective of this study was to integrate diverse variable types into a machine learning algorithm for predicting individual patient responses to CRT. METHODS: We built an ensemble classification algorithm using previously acquired data from the SMART-AV CRT clinical trial (n = 794 patients). We used five-fold stratified cross-validation on 80% of the patients (n = 635) to train the model with variables collected at 0 months (before initiating CRT), and the remaining 20% of the patients (n = 159) were used as a hold-out test set for model validation. To improve model interpretability, we quantified feature importance values using SHapley Additive exPlanations (SHAP) analysis and used Local Interpretable Model-agnostic Explanations (LIME) to explain patient-specific predictions. RESULTS: Our classification algorithm incorporated 26 patient demographic and medical history variables, 12 biomarker variables, and 18 LV functional variables, which yielded correct prediction of CRT response in 71% of patients. Additional patient stratification to identify the subgroups with the highest or lowest likelihood of response showed 96% accuracy with 22 correct predictions out of 23 patients in the highest and lowest responder groups. CONCLUSION: Computationally integrating general patient characteristics, comorbidities, therapy history, circulating biomarkers, and LV function data available before CRT intervention can improve the prediction of individual patient responses.

Ubiquitin ligase Wwp1 gene deletion attenuates diastolic dysfunction in pressure-overload hypertrophy.

Heart failure with a preserved left ventricular (LV) ejection fraction (HFpEF) often arises from a prolonged LV pressure overload (LVPO) and accompanied by abnormal extracellular matrix (ECM) accumulation. The E3 ubiquitin ligase WWP1 is a fundamental determinant ECM turnover. We tested the hypothesis that genetic ablation of Wwp1 would alter the progression of LVPO-induced HFpEF. LV echocardiography in mice with global Wwp1 deletion (n = 23; Wwp1-/-) was performed at 12 wk of age (baseline) and then at 2 and 4 wk following LVPO (transverse aortic banding) or surgery without LVPO induction. Age-matched wild-type mice (Wwp1+/+; n = 23) underwent identical protocols. LV EF remained constant and unchanged with LVPO and LV mass increased in both groups but was lower in the Wwp1-/- mice. With LVPO, the E/A ratio, an index of LV filling, was 3.97 ± 0.46 in Wwp1+/+ but was 1.73 ± 0.19 in the Wwp1-/- group (P < 0.05). At the transcriptional level, mRNA for fibrillar collagens (types I and III) decreased by approximately 50% in Wwp1-/- compared with the Wwp1+/+ group at 4 wk post-LVPO (P < 0.05) and was paralleled by a similar difference in LV fibrillar collagen content as measured by histochemistry. Moreover, mRNA levels for determinants favoring ECM accumulation, such as transforming growth factor (TGF), increased with LVPO, but were lower in the Wwp1-/- group. The absence of Wwp1 reduced the development of left ventricular hypertrophy and subsequent progression to HFpEF. Modulating the WWP1 pathway could be a therapeutic target to alter the natural history of HFpEF.NEW & NOTEWORTHY Heart failure with a preserved left ventricular (LV) ejection fraction (HFpEF) often arises from a prolonged LV pressure overload (LVPO) and is accompanied by abnormal extracellular matrix (ECM) accumulation. It is now recognized that the ECM is a dynamic entity that is regulated at multiple post-transcriptional levels, including the E3 ubiquitin ligases, such as WWP1. In the present study, WWP1 deletion in the context of an LVPO stimulus reduced functional indices of HFpEF progression and determinants of ECM remodeling.

Reperfused vs. nonreperfused myocardial infarction: when to use which model.

There is a lack of understanding in the cardiac remodeling field regarding the use of nonreperfused myocardial infarction (MI) and reperfused MI in animal models of MI. This Perspectives summarizes the consensus of the authors regarding how to select the optimum model for your experiments and is a part of ongoing efforts to establish rigor and reproducibility in cardiac physiology research.

Guidelines for in vivo mouse models of myocardial infarction.

Despite significant improvements in reperfusion strategies, acute coronary syndromes all too often culminate in a myocardial infarction (MI). The consequent MI can, in turn, lead to remodeling of the left ventricle (LV), the development of LV dysfunction, and ultimately progression to heart failure (HF). Accordingly, an improved understanding of the underlying mechanisms of MI remodeling and progression to HF is necessary. One common approach to examine MI pathology is with murine models that recapitulate components of the clinical context of acute coronary syndrome and subsequent MI. We evaluated the different approaches used to produce MI in mouse models and identified opportunities to consolidate methods, recognizing that reperfused and nonreperfused MI yield different responses. The overall goal in compiling this consensus statement is to unify best practices regarding mouse MI models to improve interpretation and allow comparative examination across studies and laboratories. These guidelines will help to establish rigor and reproducibility and provide increased potential for clinical translation.

Cytokine Signaling and Matrix Remodeling Pathways Associated with Cardiac Sarcoidosis Disease Activity Defined Using FDG PET Imaging.

While cardiac imaging has improved the diagnosis and risk assessment for cardiac sarcoidosis (CS), treatment regimens have consisted of generalized heart failure therapies and non-specific anti-inflammatory regimens. The overall goal of this study was to perform high-sensitivity plasma profiling of specific inflammatory pathways in patients with sarcoidosis and with CS.Specific inflammatory/proteolytic cascades were upregulated in sarcoidosis patients, and certain profiles emerged for CS patients.Plasma samples were collected from patients with biopsy-confirmed sarcoidosis undergoing F-18 fluorodeoxyglucose positron emission tomography (n = 47) and compared to those of referent control subjects (n = 6). Using a high-sensitivity, automated multiplex array, cytokines, soluble cytokine receptor profiles (an index of cytokine activation), as well as matrix metalloproteinase (MMP), and endogenous MMP inhibitors (TIMPs) were examined.The plasma tumor necrosis factor (TNF) and soluble TNF receptors sCD30 and sTNFRI were increased using sarcoidosis, and sTNFRII increased in CS patients (n = 18). The soluble interleukin sIL-2R and vascular endothelial growth factor receptors (sVEGFR2 and sVEGFR3) increased to the greatest degree in CS patients. When computed as a function of referent control values, the majority of soluble cytokine receptors increased in both sarcoidosis and CS groups. Plasma MMP-9 levels increased in sarcoidosis but not in the CS subset. Plasma TIMP levels declined in both groups.The findings from this study were the identification of increased activation of a cluster of soluble cytokine receptors, which augment not only inflammatory cell maturation but also transmigration in patients with sarcoidosis and patients with cardiac involvement.

The ubiquitin ligase WWP1 contributes to shifts in matrix proteolytic profiles and a myocardial aging phenotype with diastolic heart.

Ubiquitylation is a key event that regulates protein turnover, and induction of the ubiquitin ligase E3 WWP1 has been associated with age. Left ventricular hypertrophy (LVH) commonly occurs as a function of age and can cause heart failure (HF) with a preserved ejection fraction (EF; HFpEF). We hypothesized that overexpression (O/E) of WWP1 in the heart would cause LVH as well as functional and structural changes consistent with the aging HFpEF phenotype. Global WWP1 O/E was achieved in mice (n = 11) and echocardiography (40 MHz) performed to measure LV mass, EF, Doppler velocities (early E, late/atrial A), myocardial relaxation (E'), and isovolumetric relaxation time (IVRT) at 4, 6, and 8 wk. Age-matched wild-type animals (n = 15) were included as referent controls. LV EF was identical (60 ± 1 vs. 60 ± 1%, P > 0.90) with no difference in LV mass (67 ± 3 vs. 75 ± 5, P > 0.25) at 4 wk. However, at 8 wk of age, LV mass increased over twofold, E/A fell (impaired passive filling), and E/E' was lower and IVRT prolonged (impaired LV relaxation) - all P < 0.05. Collagen percent area increased by over twofold and fibrillar collagen expression (RT-PCR) over 1.5-fold (P < 0.05) with WWP1 O/E. WWP1 with an anti-WWP1 antibody could be identified in isolated cardiac fibroblasts, with WWP1 increased over twofold in O/E fibroblasts (P < 0.05). Inducing WWP1 expression caused LVH and preserved systolic function but impaired diastolic dysfunction, consistent with the HFpEF phenotype. Targeting the WWP1 pathway may be a novel therapeutic target for this intractable form of HF associated with aging.NEW & NOTEWORTHY Heart failure (HF) with a preserved ejection fraction (HFpEF) is a growing cause of HF and commonly afflicts the elderly. Milestones for HFpEF include diastolic dysfunction and an abnormal extracelluar matrix (ECM). The ubiquitin ligases, such as WWP1, change with aging and regulate critical protein turnover/stability processes, such as the ECM. The present study demonstrated that induction of WWP1 in mice induced LV hypertrophy, diastolic dysfunction, and ECM accumulation, consistent with the HFpEF phenotype, and thus may identify a new therapeutic pathway.

Targeted Injection of a Truncated Form of Tissue Inhibitor of Metalloproteinase 3 Alters Post-Myocardial Infarction Remodeling.

Infarct expansion can occur after myocardial infarction (MI), which leads to adverse left ventricular (LV) remodeling and failure. An imbalance between matrix metalloproteinase (MMP) induction and tissue inhibitors of MMPs (TIMPs) can accelerate this process. Past studies have shown different biologic effects of TIMP-3, which may depend upon specific domains within the TIMP-3 molecule. This study tested the hypothesis that differential effects of direct myocardial injections of either a full-length recombinant TIMP-3 (F-TIMP-3) or a truncated form encompassing the N-terminal region (N-TIMP-3) could be identified post-MI. MI was induced in pigs that were randomized for MI injections (30 mg) and received targeted injections within the MI region of F-TIMP-3 (n = 8), N-TIMP-3 (n = 9), or saline injection (MI-only, n = 11). At 14 days post-MI, LV ejection fraction fell post-MI but remained higher in both TIMP-3 groups. Tumor necrosis factor and interleukin-10 mRNA increased by over 10-fold in the MI-only and N-TIMP-3 groups but were reduced with F-TIMP-3 at this post-MI time point. Direct MI injection of either a full-length or truncated form of TIMP-3 is sufficient to favorably alter the course of post-MI remodeling. The functional and differential relevance of TIMP-3 domains has been established in vivo since the TIMP-3 constructs demonstrated different MMP/cytokine expression profiles. These translational studies identify a unique and more specific therapeutic strategy to alter the course of LV remodeling and dysfunction after MI. SIGNIFICANCE STATEMENT: Using different formulations of tissue inhibitor of matrix metalloproteinase-3 (TIMP-3), when injected into the myocardial infarction (MI) region, slowed the progression of indices of left ventricular (LV) failure, suggesting that the N terminus of TIMP-3 is sufficient to attenuate early adverse functional events post-MI. Injections of full-length recombinant TIMP-3, but not of the N-terminal region of TIMP-3, reduced relative indices of inflammation at the mRNA level, suggesting that the C-terminal region affects other biological pathways. These unique proof-of-concept studies demonstrate the feasibility of using recombinant small molecules to selectively interrupt adverse LV remodeling post-MI.

Focusing Heart Failure Research on Myocardial Fibrosis to Prioritize Translation.

Heart failure (HF) has traditionally been defined by symptoms of fluid accumulation and poor perfusion, but it is now recognized that specific HF classifications hold prognostic and therapeutic relevance. Specifically, HF with reduced ejection fraction is characterized by reduced left ventricular systolic pump function and dilation and HF with preserved ejection fraction is characterized primarily by abnormal left ventricular filling (diastolic failure) with relatively preserved left ventricular systolic function. These forms of HF are distributed equally among patients with HF and likely require distinctly different strategies to mitigate the morbidity, mortality, and medical resource utilization of this disease. In particular, HF is a significant medical issue within the US Department of Veterans Affairs (VA) hospital system and constitutes a major translational research priority for the VA. Because a common underpinning of both HF with reduced ejection fraction and HF with preserved ejection fraction seems to be changes in the structure and function of the myocardial extracellular matrix, a conference was convened sponsored by the VA, entitled, "Targeting Myocardial Fibrosis in Heart Failure" to explore the extracellular matrix as a potential therapeutic target and to propose specific research directions. The conference was conceptually framed around the hypothesis that although HF with reduced ejection fraction and HF with preserved ejection fraction clearly have distinct mechanisms, they may share modifiable pathways and biological mediators in common. Inflammation and extracellular matrix were identified as major converging themes. A summary of our discussion on unmet challenges and possible solutions to move the field forward, as well as recommendations for future research opportunities, are provided.

Common pathways and communication between the brain and heart: connecting post-traumatic stress disorder and heart failure.

Psychiatric illnesses and cardiovascular disease (CVD) contribute to significant overall morbidity, mortality, and health care costs, and are predicted to reach epidemic proportions with the aging population. Within the Veterans Administration (VA) health care system, psychiatric illnesses such as post-traumatic stress disorder (PTSD) and CVD such as heart failure (HF), are leading causes of hospital admissions, prolonged hospital stays, and resource utilization. Numerous studies have demonstrated associations between PTSD symptoms and CVD endpoints, particularly in the Veteran population. Not only does PTSD increase the risk of HF, but this relationship is bi-directional. Accordingly, a VA-sponsored conference entitled "Cardiovascular Comorbidities in PTSD: The Brain-Heart Consortium" was convened to explore potential relationships and common biological pathways between PTSD and HF. The conference was framed around the hypothesis that specific common systems are dysregulated in both PTSD and HF, resulting in a synergistic acceleration and amplification of both disease processes. The conference was not intended to identify all independent pathways that give rise to PTSD and HF, but rather identify shared systems, pathways, and biological mediators that would be modifiable in both disease processes. The results from this conference identified specific endocrine, autonomic, immune, structural, genetic, and physiological changes that may contribute to shared PTSD-CVD pathophysiology and could represent unique opportunities to develop therapies for both PTSD and HF. Some recommendations from the group for future research opportunities are provided.

Regional and temporal changes in left ventricular strain and stiffness in a porcine model of myocardial infarction.

The aim of the present study was to serially track how myocardial infarction (MI) impacts regional myocardial strain and mechanical properties of the left ventricle (LV) in a large animal model. Post-MI remodeling has distinct regional effects throughout the LV myocardium. Regional quantification of LV biomechanical behavior could help explain changes in global function and thus advance clinical assessment of post-MI remodeling. The present study is based on a porcine MI model to characterize LV biomechanics over 28 days post-MI via speckle-tracking echocardiography (STE). Regional myocardial strain and strain rate were recorded in the circumferential, radial, and longitudinal directions at baseline and at 3, 14, and 28 days post-MI. Regional myocardial wall stress was calculated using standard echocardiographic metrics of geometry and Doppler-derived hemodynamic measurements. Regional diastolic myocardial stiffness was calculated from the resultant stress-strain relations. Peak strain and phasic strain rates were nonuniformly reduced throughout the myocardium post-MI, whereas time to peak strain was increased to a similar degree in the MI region and border zone by 28 days post-MI. Elevations in diastolic myocardial stiffness in the MI region plateaued at 14 days post-MI, after which a significant reduction in MI regional stiffness in the longitudinal direction occurred between 14 and 28 days post-MI. Post-MI biomechanical changes in the LV myocardium were initially limited to the MI region but nonuniformly extended into the neighboring border zone and remote myocardium over 28 days post-MI. STE enabled quantification of regional and temporal differences in myocardial strain and diastolic stiffness, underscoring the potential of this technique for clinical assessment of post-MI remodeling. NEW & NOTEWORTHY For the first time, speckle-tracking echocardiography was used to serially track regional biomechanical behavior and mechanical properties postmyocardial infarction (post-MI). We found that changes initially confined to the MI region extended throughout the myocardium in a nonuniform fashion over 28 days post-MI. Speckle-tracking echocardiography-based evaluation of regional changes in left ventricular biomechanics could advance both clinical assessment of left ventricular remodeling and therapeutic strategies that target aberrant biomechanical behavior post-MI.

Identification of a novel microRNA profile in pediatric patients with cancer treated with anthracycline chemotherapy.

Anthracycline chemotherapy (AC) is associated with decline in left ventricular ejection fraction (LVEF), yet the mechanisms remain unclear. Although changes in microRNAs (miRs) have been identified in adult cardiovascular disease, miR profiles in pediatric patients with AC have not been well studied. The goal of this study was to examine miR profiles (unbiased array) in pediatric patients with AC compared with age-matched referent normal patients. We hypothesize that pediatric patients with AC will express a unique miR profile at the initiation and completion of therapy and will be related to LVEF. Serum was collected in pediatric patients (10-22 yr, n = 12) with newly diagnosed malignancy requiring AC within 24-48 h after the initiation of therapy (30-60 mg/m2) and ~1 yr after completing therapy. A custom microarray of 84 miRs associated with cardiovascular disease was used (quantitative RT-PCR) and indexed to referent normal profiles (13-17 yr, n = 17). LVEF was computed by cardiac MRI. LVEF fell from AC initiation at ~1 yr after AC completion (64.28 ± 1.78% vs. 57.53 ± 0.95%, respectively, P = 0.004). Of the 84 miRs profiled, significant shifts in 17 miRs occurred relative to referent normal ( P ≤ 0.05). Moreover, the functional domain of miRs associated with myocardial differentiation and development fell over threefold at the completion of AC ( P ≤ 0.05). Moreover, eight miRs were significantly downregulated after AC completion in those patients with the greatest decline in LVEF (≥10%, P < 0.05). This study demonstrates, for the first time, that changes in miR expression occur in pediatric patients with AC. These findings suggest that miRs are a potential strategy for the early identification of patients with AC susceptible to left ventricular dysfunction. NEW & NOTEWORTHY Although anthracycline chemotherapy (AC) is effective for a number of pediatric cancers, an all too often consequence of AC is the development of left ventricular failure. The present study identified that specific shifts in the pattern of microRNAs, which regulate myocardial growth, function, and viability, occurred during and after AC in pediatric patients, whereby the magnitude of this shift was associated with the degree of left ventricular failure.

Delivery of a matrix metalloproteinase-responsive hydrogel releasing TIMP-3 after myocardial infarction: effects on left ventricular remodeling.

Although improvements in timing and approach for early reperfusion with acute coronary syndromes have occurred, myocardial injury culminating in a myocardial infarction (MI) remains a common event. Although a multifactorial process, an imbalance between the induction of proteolytic pathways, such as matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of metalloproteinase (TIMPs), has been shown to contribute to this process. In the present study, a full-length TIMP-3 recombinant protein (rTIMP-3) was encapsulated in a specifically formulated hyaluronic acid (HA)-based hydrogel that contained MMP-cleavable peptide cross-links, which influenced the rate of rTIMP-3 release from the HA gel. The effects of localized delivery of this MMP-sensitive HA gel (HAMMPS) alone and containing rTIMP-3 (HAMMPS/rTIMP-3) were examined in terms of the natural history of post-MI remodeling. Pigs were randomized to one of the following three different groups: MI and saline injection (MI/saline group, 100-µl injection at nine injection sites, n = 7), MI and HAMMPS injection (MI/HAMMPS group; 100-µl injection at nine injection sites, n = 7), and MI and HAMMPS/rTIMP-3 injection (MI/HAMMPS/rTIMP-3 group; 20-µg/100-µl injection at nine injection sites, n = 7). Left ventricular (LV) echocardiography was serially performed up to 28 days post-MI. LV dilation, as measured by end-diastolic volume, and the degree of MI wall thinning were reduced by ~50% in the HAMMPS/rTIMP-3 group ( P < 0.05). Furthermore, indexes of heart failure progression post-MI, such as LV filling pressures and left atrial size, were also attenuated to the greatest degree in the HAMMPS/rTIMP-3 group. At 28 days post-MI, HAMMPS/rTIMP-3 caused a relative reduction in the transcriptional profile for myofibroblasts as well as profibrotic pathways, which was confirmed by subsequent histochemistry. In conclusion, these findings suggest that localized delivery of a MMP-sensitive biomaterial that releases a recombinant TIMP holds promise as a means to interrupt adverse post-MI remodeling. NEW & NOTEWORTHY The present study targeted a myocardial matrix proteolytic system, matrix metalloproteinases (MMPs), through the use of a recombinant tissue inhibitor of MMPs incorporated into a MMP-sensitive hydrogel, which was regionally injected using a large animal model of myocardial infarction. Left ventricular geometry and function and indexes of myocardial remodeling were improved with this approach and support the advancement of localized therapeutic strategies that specifically target the myocardial matrix.

Contractile Smooth Muscle and Active Stress Generation in Porcine Common Carotids.

The mechanical response of intact blood vessels to applied loads can be delineated into passive and active components using an isometric decomposition approach. Whereas the passive response is due predominantly to the extracellular matrix (ECM) proteins and amorphous ground substance, the active response depends on the presence of smooth muscle cells (SMCs) and the contractile machinery activated within those cells. To better understand determinants of active stress generation within the vascular wall, we subjected porcine common carotid arteries (CCAs) to biaxial inflation-extension testing under maximally contracted or passive SMC conditions and semiquantitatively measured two known markers of the contractile SMC phenotype: smoothelin and smooth muscle-myosin heavy chain (SM-MHC). Using isometric decomposition and established constitutive models, an intuitive but novel correlation between the magnitude of active stress generation and the relative abundance of smoothelin and SM-MHC emerged. Our results reiterate the importance of stretch-dependent active stress generation to the total mechanical response. Overall these findings can be used to decouple the mechanical contribution of SMCs from the ECM and is therefore a powerful tool in the analysis of disease states and potential therapies where both constituent are altered.

Intracoronary delivery of recombinant TIMP-3 after myocardial infarction: effects on myocardial remodeling and function.

Ischemia-reperfusion (IR) and myocardial infarction (MI) cause adverse left ventricular (LV) remodeling and heart failure and are facilitated by an imbalance in matrix metalloproteinase (MMP) activation and the endogenous tissue inhibitors of metalloproteinase (TIMPs). We have identified that myocardial injections of recombinant TIMP-3 (rTIMP-3; human full length) can interrupt post-MI remodeling. However, whether and to what degree intracoronary delivery of rTIMP-3 post-IR is feasible and effective remained to be established. Pigs (25 kg) underwent coronary catheterization and balloon occlusion of the left anterior descending coronary artery (LAD) for 90 min whereby at the final 4 min, rTIMP-3 (30 mg, n = 9) or saline was infused in the distal LAD. LV echocardiography was performed at 3-28 days post-IR, and LV ejection fraction (EF) and LV end-diastolic volume were measured. LV EF fell and LV end-diastolic volume increased from baseline (pre-IR) values (66 ± 1% and 40 ± 1 ml, respectively, means ± standard deviation) in both groups; however, the extent of LV dilation was reduced in the rTIMP-3 group by 40% at 28 days post-IR (P < 0.05) and the fall in LV EF was attenuated. Despite equivalent plasma troponin levels (14 ± 3 ng/ml), computed MI size at 28 days was reduced by over 45% in the rTIMP-3 group (P < 0.05), indicating that rTIMP-3 treatment abrogated MI expansion post-IR. Plasma NH2-terminal pro-brain natriuretic peptide levels, an index of heart failure progression, were reduced by 25% in the rTIMP-3 group compared with MI saline values (P < 0.05). Although the imbalance between MMPs and TIMPs has been recognized as a contributory factor for post-MI remodeling, therapeutic strategies targeting this imbalance have not been forthcoming. This study is the first to demonstrate that a relevant delivery approach (intracoronary) using rTIMP can alter the course of post-MI remodeling.NEW & NOTEWORTHY Myocardial ischemia and reperfusion injury remain significant causes of morbidity and mortality whereby alterations in the balance between matrix metalloproteinase and tissue inhibitor of metalloproteinase have been identified as contributory biological mechanisms. This novel translational study advances the concept of targeted delivery of recombinant proteins to modify adverse myocardial remodeling in ischemia-reperfusion injury.

Basigin Promotes Cardiac Fibrosis and Failure in Response to Chronic Pressure Overload in Mice.

OBJECTIVE: Basigin (Bsg) is a transmembrane glycoprotein that activates matrix metalloproteinases and promotes inflammation. However, the role of Bsg in the pathogenesis of cardiac hypertrophy and failure remains to be elucidated. We examined the role of Bsg in cardiac hypertrophy and failure in mice and humans. APPROACH AND RESULTS: We performed transverse aortic constriction in Bsg(+/-) and in wild-type mice. Bsg(+/-) mice showed significantly less heart and lung weight and cardiac interstitial fibrosis compared with littermate controls after transverse aortic constriction. Both matrix metalloproteinase activities and oxidative stress in loaded left ventricle were significantly less in Bsg(+/-) mice compared with controls. Echocardiography showed that Bsg(+/-) mice showed less hypertrophy, less left ventricular dilatation, and preserved left ventricular fractional shortening compared with littermate controls after transverse aortic constriction. Consistently, Bsg(+/-) mice showed a significantly improved long-term survival after transverse aortic constriction compared with Bsg(+/+) mice, regardless of the source of bone marrow (Bsg(+/+) or Bsg(+/-)). Conversely, cardiac-specific Bsg-overexpressing mice showed significantly poor survival compared with littermate controls. Next, we isolated cardiac fibroblasts and examined their responses to angiotensin II or mechanical stretch. Both stimuli significantly increased Bsg expression, cytokines/chemokines secretion, and extracellular signal-regulated kinase/Akt/JNK activities in Bsg(+/+) cardiac fibroblasts, all of which were significantly less in Bsg(+/-) cardiac fibroblasts. Consistently, extracellular and intracellular Bsg significantly promoted cardiac fibroblast proliferation. Finally, serum levels of Bsg were significantly elevated in patients with heart failure and predicted poor prognosis. CONCLUSIONS: These results indicate the crucial roles of intracellular and extracellular Bsg in the pathogenesis of cardiac hypertrophy, fibrosis, and failure in mice and humans.

Targeted overexpression of tissue inhibitor of matrix metalloproteinase-4 modifies post-myocardial infarction remodeling in mice.

RATIONALE: Myocardial infarction (MI) causes an imbalance between matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases (TIMPs) and is associated with adverse left ventricular (LV) remodeling. A uniform reduction in TIMP-4 post-MI has been observed. OBJECTIVE: To examine post-MI remodeling with cardiac-restricted overexpression of TIMP-4, either through a transgenic or viral delivery approach. METHODS AND RESULTS: MI was induced in mice and then randomized to targeted injection of an adenoviral construct (10 µL; 8×10(9) plaque forming units/mL) encoding green fluorescent protein (GFP) and the full-length human TIMP-4 (Ad-GFP-TIMP4) or GFP. A transgenic construct with cardiac-restricted overexpression TIMP-4 (hTIMP-4exp) was used in a parallel set of studies. LV end-diastolic volume, an index of LV remodeling, increased by >60% from baseline at 5 days post-MI and by >100% at 21 days post-MI in the Ad-GFP only group. However, LV dilation was reduced by ≈50% in both the Ad-GFP-TIMP4 and hTIMP-4exp groups at these post-MI time points. LV ejection fraction was improved with either Ad-GFP-TIMP-4 or hTIMP-4exp. Fibrillar collagen expression and content were increased within the MI region with both TIMP-4 interventions, suggestive of matrix stabilization. CONCLUSIONS: This study is the first to demonstrate that selective myocardial targeting for TIMP-4 induction through either a viral or transgenic approach favorably altered the course of adverse LV remodeling post-MI. Thus, localized induction of endogenous matrix metalloproteinase inhibitors, such as TIMP-4, holds promise as a means to interrupt the progression of post-MI remodeling.

Inhibition of class I histone deacetylase activity represses matrix metalloproteinase-2 and -9 expression and preserves LV function postmyocardial infarction.

Left ventricular (LV) remodeling, after myocardial infarction (MI), can result in LV dilation and LV pump dysfunction. Post-MI induction of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated as causing deleterious effects on LV and extracellular matrix remodeling in the MI region and within the initially unaffected remote zone. Histone deacetylases (HDACs) are a class of enzymes that affect the transcriptional regulation of genes during pathological conditions. We assessed the efficacy of both class I/IIb- and class I-selective HDAC inhibitors on MMP-2 and MMP-9 abundance and determined if treatment resulted in the attenuation of adverse LV and extracellular matrix remodeling and improved LV pump function post-MI. MI was surgically induced in MMP-9 promoter reporter mice and randomized for treatment with a class I/IIb HDAC inhibitor for 7 days post-MI. After MI, LV dilation, LV pump dysfunction, and activation of the MMP-9 gene promoter were significantly attenuated in mice treated with either the class I/IIb HDAC inhibitor tichostatin A or suberanilohydroxamic acid (voronistat) compared with MI-only mice. Immunohistological staining and zymographic levels of MMP-2 and MMP-9 were reduced with either tichostatin A or suberanilohydroxamic acid treatment. Class I HDAC activity was dramatically increased post-MI. Treatment with the selective class I HDAC inhibitor PD-106 reduced post-MI levels of both MMP-2 and MMP-9 and attenuated LV dilation and LV pump dysfunction post-MI, similar to class I/IIb HDAC inhibition. Taken together, these unique findings demonstrate that selective inhibition of class I HDACs may provide a novel therapeutic means to attenuate adverse LV remodeling post-MI.

Injectable and bioresponsive hydrogels for on-demand matrix metalloproteinase inhibition.

Inhibitors of matrix metalloproteinases (MMPs) have been extensively explored to treat pathologies where excessive MMP activity contributes to adverse tissue remodelling. Although MMP inhibition remains a relevant therapeutic target, MMP inhibitors have not translated to clinical application owing to the dose-limiting side effects following systemic administration of the drugs. Here, we describe the synthesis of a polysaccharide-based hydrogel that can be locally injected into tissues and releases a recombinant tissue inhibitor of MMPs (rTIMP-3) in response to MMP activity. Specifically, rTIMP-3 is sequestered in the hydrogels through electrostatic interactions and is released as crosslinks are degraded by active MMPs. Targeted delivery of the hydrogel/rTIMP-3 construct to regions of MMP overexpression following a myocardial infarction significantly reduced MMP activity and attenuated adverse left ventricular remodelling in a porcine model of myocardial infarction. Our findings demonstrate that local, on-demand MMP inhibition is achievable through the use of an injectable and bioresponsive hydrogel.