Alison L. Müller

Role of various proteases in cardiac remodeling and progression of heart failure

It is believed that cardiac remodeling due to geometric and structural changes is a major mechanism for the progression of heart failure in different pathologies including hypertension, hypertrophic cardiomyopathy, dilated cardiomyopathy, diabetic cardiomyopathy, and myocardial infarction. Increases in the activities of proteolytic enzymes such as matrix metalloproteinases, calpains, cathepsins, and caspases contribute to the process of cardiac remodeling. In addition to modifying the extracellular matrix, both matrix metalloproteinases and cathepsins have been shown to affect the activities of subcellular organelles in cardiomyocytes. The activation of calpains and caspases has been identified to induce subcellular remodeling in failing hearts. Proteolytic activities associated with different proteins including caspases, calpain, and the ubiquitin–proteasome system have been shown to be involved in cardiomyocyte apoptosis, which is an integral part of cardiac remodeling. This article discusses and compares how the activities of various proteases are involved in different cardiac abnormalities with respect to alterations in apoptotic pathways, cardiac remodeling, and cardiac dysfunction. An imbalance appears to occur between the activities of some proteases and their endogenous inhibitors in various types of hypertrophied and failing hearts, and this is likely to further accentuate subcellular remodeling and cardiac dysfunction. The importance of inhibiting the activities of both extracellular and intracellular proteases specific to distinct etiologies, in attenuating cardiac remodeling and apoptosis as well as biochemical changes of subcellular organelles, in heart failure has been emphasized. It is suggested that combination therapy to inhibit different proteases may prove useful for the treatment of heart failure.

Extracellular and intracellular proteases in cardiac dysfunction due to ischemia-reperfusion injury

Various procedures such as angioplasty, thrombolytic therapy, coronary bypass surgery, and cardiac transplantation are invariably associated with ischemia-reperfusion (I/R) injury. Impaired recovery of cardiac function due to I/R injury is considered to be a consequence of the occurrence of both oxidative stress and intracellular Ca(2+)-overload in the myocardium. These changes in the ischemic myocardium appear to activate both extracellular and intracellular proteases which are responsible for the cleavage of extracellular matrix and subcellular structures involved in the maintenance of cardiac function. It is thus intended to discuss the actions of I/R injury on several proteases, with a focus on calpain, matrix metalloproteinases, and cathepsins as well as their role in inducing alterations both inside and outside the cardiomyocytes. In addition, modifications of subcellular organelles such as myofibrils, sarcoplasmic reticulum and sarcolemma as well as extracellular matrix, and the potential regulatory effects of endogenous inhibitors on protease activities are identified. Both extracellular and intracellular proteolytic activities appear to be imperative in determining the true extent of I/R injury and their inhibition seems to be of critical importance for improving the recovery of cardiac function. Thus, both extracellular and intracellular proteases may serve as potential targets for the development of cardioprotective interventions for reducing damage to the heart and retarding the development of contractile dysfunction caused by I/R injury.

A whole blood–based perfusate provides superior preservation of myocardial function during ex vivo heart perfusion

BACKGROUND Ex vivo heart perfusion (EVHP) provides the opportunity to resuscitate unused donor organs and facilitates assessments of myocardial function that are required to demonstrate organ viability before transplantation. We sought to evaluate the effect of different oxygen carriers on the preservation of myocardial function during EVHP. METHODS Twenty-seven pig hearts were perfused ex vivo in a normothermic beating state for 6 hours and transitioned into working mode for assessments after 1 (T1), 3 (T3), and 5 (T5) hours. Hearts were allocated to 4 groups according to the perfusate composition. Red blood cell concentrate (RBC, n = 6), whole blood (RBC+Plasma, n = 6), an acellular hemoglobin-based oxygen carrier (HBOC, n = 8), or HBOC plus plasma (HBOC+Plasma, n = 7) were added to STEEN Solution (XVIVO Perfusion, Goteborg, Sweden) to achieve a perfusate hemoglobin concentration of 40 g/liter. RESULTS The perfusate composition affected the preservation of systolic (T5 dP/dtmax: RBC+Plasma = 903 ± 99, RBC = 771 ± 77, HBOC+Plasma = 691 ± 82, HBOC = 563 ± 52 mm Hg/sec; p = 0.047) and diastolic (T5 dP/dtmin: RBC+Plasma = -574 ± 48, RBC = -492 ± 63, HBOC+Plasma = -326 ± 32, HBOC = -268 ± 22 mm Hg/sec; p < 0.001) function, and the development of myocardial edema (weight gain: RBC+Plasma = 6.6 ± 0.9, RBC = 6.6 ± 1.2, HBOC+Plasma = 9.8 ± 1.7, HBOC = 16.3 ± 1.9 g/hour; p < 0.001) during EVHP. RBC+Plasma hearts exhibited less histologic evidence of myocyte damage (injury score: RBC+Plasma = 0.0 ± 0.0, RBC = 0.8 ± 0.3, HBOC+Plasma = 2.6 ± 0.2, HBOC = 1.75 ± 0.4; p < 0.001) and less troponin-I release (troponin-I fold-change T1-T5: RBC+Plasma = 7.0 ± 1.7, RBC = 13.1 ± 1.6, HBOC+Plasma = 20.5 ± 1.1, HBOC = 16.7 ± 5.8; p < 0.001). Oxidative stress was minimized by the addition of plasma to RBC and HBOC hearts (oxidized phosphatidylcholine compound fold-change T1-T5: RBC+Plasma = 1.83 ± 0.20 vs RBC = 2.31 ± 0.20, p < 0.001; HBOC+Plasma = 1.23 ± 0.17 vs HBOC = 2.80 ± 0.28, p < 0.001). CONCLUSIONS A whole blood-based perfusate (RBC+Plasma) minimizes injury and provides superior preservation of myocardial function during EVHP. The beneficial effect of plasma on the preservation of myocardial function requires further investigation.

Human mesenchymal stem cells express a myofibroblastic phenotype in vitro: comparison to human cardiac myofibroblasts

Abstract Cardiac fibrosis accompanies a variety of myocardial disorders, and is induced by myofibroblasts. These cells may be composed of a heterogeneous population of parent cells, including interstitial fibroblasts and circulating progenitor cells. Direct comparison of human bone marrow-derived mesenchymal stem cells (BM-MSCs) and cardiac myofibroblasts (CMyfbs) has not been previously reported. We hypothesized that BM-MSCs readily adopt a myofibroblastic phenotype in culture. Human primary BM-MSCs and human CMyfbs were isolated from patients undergoing open heart surgery and expanded under standard culture conditions. We assessed and compared their phenotypic and functional characteristics by examining their gene expression profile, their ability to contract collagen gels and synthesize collagen type I. In addition, we examined the role of non-muscle myosin II (NMMII) in modulating MSC myogenic function using NMMII siRNA knockdown and blebbistatin, a specific small molecule inhibitor of NMMII. We report that, while human BM-MSCs retain pluripotency, they adopt a myofibroblastic phenotype in culture and stain positive for the myofibroblast markers α-SMA, vimentin, NMMIIB, ED-A fibronectin, and collagen type 1 at each passage. In addition, they contract collagen gels in response to TGF-β1 and synthesize collagen similar to human CMyfbs. Moreover, inhibition of NMMII activity with blebbistatin completely attenuates gel contractility without affecting cell viability. Thus, human BM-MSCs share and exhibit similar physiological and functional characteristics as human CMyfbs in vitro, and their propensity to adopt a myofibroblast phenotype in culture may contribute to cardiac fibrosis.

Mechanisms of the Beneficial Actions of Ischemic Preconditioning on Subcellular Remodeling in Ischemic-Reperfused Heart

Cardiac function is compromised by oxidative stress which occurs upon exposing the heart to ischemia reperfusion (I/R) for a prolonged period. The reactive oxygen species (ROS) that are generated during I/R incur extensive damage to the myocardium and result in subcellular organelle remodeling. The cardiac nucleus, glycocalyx, myofilaments, sarcoplasmic reticulum, sarcolemma, and mitochondria are affected by ROS during I/R injury. On the other hand, brief periods of ischemia followed by reperfusion, or ischemic preconditioning (IPC), have been shown to be cardioprotective against oxidative stress by attenuating the cellular damage and alterations of subcellular organelles caused by subsequent I/R injury. Endogenous defense mechanisms, such as antioxidant enzymes and heat shock proteins, are activated by IPC and thus prevent damage caused by oxidative stress. Although these cardioprotective effects of IPC against I/R injury are considered to be a consequence of changes in the redox state of cardiomyocytes, IPC is considered to promote the production of NO which may protect subcellular organelles from the deleterious actions of oxidative stress. The article is intended to focus on the I/R-induced oxidative damage to subcellular organelles and to highlight the cardioprotective effects of IPC. In addition, the actions of various endogenous cardioprotective interventions are discussed to illustrate that changes in the redox state due to IPC are cardioprotective against I/R injury to the heart.

Protein Kinases as Drug Development Targets for Heart Disease Therapy

Protein kinases are intimately integrated in different signal transduction pathways for the regulation of cardiac function in both health and disease. Protein kinase A (PKA), Ca2+-calmodulin-dependent protein kinase (CaMK), protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) are not only involved in the control of subcellular activities for maintaining cardiac function, but also participate in the development of cardiac dysfunction in cardiac hypertrophy, diabetic cardiomyopathy, myocardial infarction, and heart failure. Although all these kinases serve as signal transducing proteins by phosphorylating different sites in cardiomyocytes, some of their effects are cardioprotective whereas others are detrimental. Such opposing effects of each signal transduction pathway seem to depend upon the duration and intensity of stimulus as well as the type of kinase isoform for each kinase. In view of the fact that most of these kinases are activated in heart disease and their inhibition has been shown to improve cardiac function, it is suggested that these kinases form excellent targets for drug development for therapy of heart disease.

Physiologic Changes in the Heart Following Cessation of Mechanical Ventilation in a Porcine Model of Donation After Circulatory Death: Implications for Cardiac Transplantation.

Hearts donated following circulatory death (DCD) may represent an additional source of organs for transplantation; however, the impact of donor extubation on the DCD heart has not been well characterized. We sought to describe the physiologic changes that occur following withdrawal of life‐sustaining therapy (WLST) in a porcine model of DCD. Physiologic changes were monitored continuously for 20 min following WLST. Ventricular pressure, volume, and function were recorded using a conductance catheter placed into the right (N = 8) and left (N = 8) ventricles, and using magnetic resonance imaging (MRI, N = 3). Hypoxic pulmonary vasoconstriction occurred following WLST, and was associated with distension of the right ventricle (RV) and reduced cardiac output. A 120‐fold increase in epinephrine was subsequently observed that produced a transient hyperdynamic phase; however, progressive RV distension developed during this time. Circulatory arrest occurred 7.6±0.3 min following WLST, at which time MRI demonstrated an 18±7% increase in RV volume and a 12±9% decrease in left ventricular volume compared to baseline. We conclude that hypoxic pulmonary vasoconstriction and a profound catecholamine surge occur following WLST that result in distension of the RV. These changes have important implications on the resuscitation, preservation, and evaluation of DCD hearts prior to transplantation.

Activation of proteases and changes in Na+-K+-ATPase subunits in hearts subjected to ischemia-reperfusion

Previous studies have shown that ischemia-reperfusion (I/R) injury is associated with cardiac dysfunction and changes in sarcolemmal Na(+)-K(+)-ATPase subunits and activity. This study was undertaken to evaluate the role of proteases in these alterations by subjecting rat hearts to different times of global ischemia, as well as reperfusion after 45 min of ischemia. Decreases in Na(+)-K(+)-ATPase activity at 30-60 min of global ischemia were accompanied by augmented activities of both calpain and matrix metalloproteinases (MMPs) and depressed protein content of β(1)- and β(2)-subunits, without changes in α(1)- and α(2)-subunits of the enzyme. Compared with control values, the activities of both calpain and MMP-2 were increased, whereas the activity and protein content for all subunits of Na(+)-K(+)-ATPase were decreased upon reperfusion for 5-40 min, except that α(1)- and α(2)-subunit content was not depressed in 5 min I/R hearts. MDL28170, a calpain inhibitor, was more effective in attenuating the I/R-induced alterations in cardiac contracture, Na(+)-K(+)-ATPase activity, and α(2)-subunit than doxycycline, an MMP inhibitor. Incubation of control sarcolemma preparation with calpain, unlike MMP-2, depressed Na(+)-K(+)-ATPase activity and decreased α(1)-, α(2)-, and β(2)-subunits, without changes in the β(1)-subunit. These results support the view that activation of both calpain and MMP-2 are involved in depressing Na(+)-K(+)-ATPase activity and degradation of its subunits directly or indirectly in hearts subjected to I/R injury.

Avoidance of Profound Hypothermia During Initial Reperfusion Improves the Functional Recovery of Hearts Donated After Circulatory Death

The resuscitation of hearts donated after circulatory death (DCD) is gaining widespread interest; however, the method of initial reperfusion (IR) that optimizes functional recovery has not been elucidated. We sought to determine the impact of IR temperature on the recovery of myocardial function during ex vivo heart perfusion (EVHP). Eighteen pigs were anesthetized, mechanical ventilation was discontinued, and cardiac arrest ensued. A 15‐min standoff period was observed and then hearts were reperfused for 3 min at three different temperatures (5°C; N = 6, 25°C; N = 5, and 35°C; N = 7) with a normokalemic adenosine–lidocaine crystalloid cardioplegia. Hearts then underwent normothermic EVHP for 6 h during which time myocardial function was assessed in a working mode. We found that IR coronary blood flow differed among treatment groups (5°C = 483 ± 53, 25°C = 722 ± 60, 35°C = 906 ± 36 mL/min, p < 0.01). During subsequent EVHP, less myocardial injury (troponin I: 5°C = 91 ± 6, 25°C = 64 ± 16, 35°C = 57 ± 7 pg/mL/g, p = 0.04) and greater preservation of endothelial cell integrity (electron microscopy injury score: 5°C = 3.2 ± 0.5, 25°C = 1.8 ± 0.2, 35°C = 1.7 ± 0.3, p = 0.01) were evident in hearts initially reperfused at warmer temperatures. IR under profoundly hypothermic conditions impaired the recovery of myocardial function (cardiac index: 5°C = 3.9 ± 0.8, 25°C = 6.2 ± 0.4, 35°C = 6.5 ± 0.6 mL/minute/g, p = 0.03) during EVHP. We conclude that the avoidance of profound hypothermia during IR minimizes injury and improves the functional recovery of DCD hearts.

Implications of protease activation in cardiac dysfunction and development of genetic cardiomyopathy in hamsters

It has become evident that protein degradation by proteolytic enzymes, known as proteases, is partly responsible for cardiovascular dysfunction in various types of heart disease. Both extracellular and intracellular alterations in proteolytic activities are invariably seen in heart failure associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy. Genetic cardiomyopathy displayed in different strains of hamsters provides a useful model for studying heart failure due to either cardiac hypertrophy or cardiac dilation. Alterations in the function of several myocardial organelles such as sarcolemma, sarcoplasmic reticulum, myofibrils, mitochondria, as well as extracellular matrix have been shown to be due to subcellular remodeling as a consequence of changes in gene expression and protein content in failing hearts from cardiomyopathic hamsters. In view of the increased activities of various proteases, including calpains and matrix metalloproteinases in the hearts of genetically determined hamsters, it is proposed that the activation of different proteases may also represent an important determinant of subcellular remodeling and cardiac dysfunction associated with genetic cardiomyopathy.