Sarah Williams

Perforin-Independent Extracellular Granzyme B Activity Contributes to Abdominal Aortic Aneurysm

Granzyme B (GZMB) is a serine protease that is abundantly expressed in advanced human atherosclerotic lesions and may contribute to plaque instability. Perforin is a pore-forming protein that facilitates GZMB internalization and the induction of apoptosis. Recently a perforin-independent, extracellular role for GZMB has been proposed. In the current study, the role of GZMB in abdominal aortic aneurysm (AAA) was assessed. Apolipoprotein E (APOE)(-/-) x GZMB(-/-) and APOE(-/-) x perforin(-/-) double knockout (GDKO, PDKO) mice were generated to test whether GZMB exerted a causative role in aneurysm formation. To induce aneurysm, mice were given angiotensin II (1000 ng/kg/min) for 28 days. GZMB was found to be abundant in both murine and human AAA specimens. GZMB deficiency was associated with a decrease in AAA and increased survival compared with APOE-KO and PDKO mice. Although AAA rupture was observed frequently in APOE-KO (46.7%; n = 15) and PDKO (43.3%; n = 16) mice, rupture was rarely observed in GDKO (7.1%; n = 14) mice. APOE-KO mice exhibited reduced fibrillin-1 staining compared with GDKO mice, whereas in vitro protease assays demonstrated that fibrillin-1 is a substrate of GZMB. As perforin deficiency did not affect the outcome, our results suggest that GZMB contributes to AAA pathogenesis via a perforin-independent mechanism involving extracellular matrix degradation and subsequent loss of vessel wall integrity.

Granzymes in age-related cardiovascular and pulmonary diseases

Chronic inflammation is a hallmark of age-related cardiovascular and pulmonary diseases. Granzymes are a family of serine proteases that have been traditionally viewed as initiators of immune-mediated cell death. However, recent findings suggest that the pathophysiological role of granzymes is complex. Emerging functions for granzymes in extracellular matrix degradation, autoimmunity, and inflammation suggests a multifactorial mechanism by which these enzymes are capable of mediating tissue damage. Recent discoveries showing that granzymes can be produced and secreted by nonimmune cells during disease provide an additional layer of intricacy. This review examines the emerging biochemical and clinical evidence pertaining to intracellular and/or extracellular granzymes in the pathogenesis of aging and cardiopulmonary diseases.

Regulation of TRPV2 by axotomy in sympathetic, but not sensory neurons.

Neuropathic pain results from traumatic or disease-related insults to the nervous system. Mechanisms that have been postulated to underlie peripheral neuropathy commonly implicate afferent neurons that have been damaged but still project centrally to the spinal cord, and/or intact neurons that interact with degenerating distal portions of the injured neurons. One pain state that is observed following peripheral nerve injury in the rat is thermal hyperalgesia. The noxious heat-gated ion channel TRPV1 may be responsible for this increased sensitivity, as it is up-regulated in L4 dorsal root ganglion (DRG) neurons following L5 spinal nerve lesion (SpNL). The TRPV1 homologue TRPV2 (or VRL-1) is another member of the TRPV subfamily of TRP ion channels. TRPV2 is a nonselective cation channel activated by high noxious temperatures (>52 degrees C) and is present in a subset of medium- to large-diameter DRG neurons. To establish whether TRPV2 is endogenous to the spinal cord, we examined its expression in the dorsal horn following rhizotomy. We found no significant decrease in TRPV2 immunoreactivity, suggesting that TRPV2 is endogenous to the spinal cord. In order to determine whether TRPV2, like TRPV1, is regulated by peripheral axotomy, we performed L5 SpNL and characterized TRPV2 distribution in the DRG, spinal cord, brainstem, and sympathetic ganglia. Our results show that peripheral axotomy did not regulate TRPV2 in the DRG, spinal cord, or brainstem; however, TRPV2 was up-regulated in sympathetic postganglionic neurons following injury, suggesting a potential role for TRPV2 in sympathetically mediated neuropathic pain.

Ranolazine improves diastolic function in spontaneously hypertensive rats

Diastolic dysfunction can lead to heart failure with preserved ejection fraction, for which there is no effective therapeutic. Ranolazine has been reported to reduce diastolic dysfunction, but the specific mechanisms of action are unclear. The effect of ranolazine on diastolic function was examined in spontaneously hypertensive rats (SHRs), where left ventricular relaxation is impaired and stiffness increased. The objective of this study was to determine whether ranolazine improves diastolic function in SHRs and identify the mechanism(s) by which improvement is achieved. Specifically, to test the hypothesis that ranolazine, by inhibiting late sodium current, reduces Ca(2+) overload and promotes ventricular relaxation and reduction in diastolic stiffness, the effects of ranolazine or vehicle on heart function and the response to dobutamine challenge were evaluated in aged male SHRs and Wistar-Kyoto rats by echocardiography and pressure-volume loop analysis. The effects of ranolazine and the more specific sodium channel inhibitor tetrodotoxin were determined on the late sodium current, sarcomere length, and intracellular calcium in isolated cardiomyocytes. Ranolazine reduced the end-diastolic pressure-volume relationship slope and improved diastolic function during dobutamine challenge in the SHR. Ranolazine and tetrodotoxin also enhanced cardiomyocyte relaxation and reduced myoplasmic free Ca(2+) during diastole at high-stimulus rates in the SHR. The density of the late sodium current was elevated in SHRs. In conclusion, ranolazine was effective in reducing diastolic dysfunction in the SHR. Its mechanism of action, at least in part, is consistent with inhibition of the increased late sodium current in the SHR leading to reduced Ca(2+) overload.

Granzyme B Deficiency Protects against Angiotensin II–Induced Cardiac Fibrosis

Cardiac fibrosis is observed across diverse etiologies of heart failure. Granzyme B (GzmB) is a serine protease involved in cell-mediated cytotoxicity in conjunction with the pore-forming protein, perforin. Recent evidence suggests that GzmB also contributes to matrix remodeling and fibrosis through an extracellular, perforin-independent process. However, the role of GzmB in the onset and progression of cardiac fibrosis remains elusive. The present study investigated the role of GzmB in the pathogenesis of cardiac fibrosis. GzmB was elevated in fibrotic human hearts and in angiotensin II-induced murine cardiac fibrosis. Genetic deficiency of GzmB reduced angiotensin II-induced cardiac hypertrophy and fibrosis, independently of perforin. GzmB deficiency also reduced microhemorrhage, inflammation, and fibroblast accumulation inxa0vivo. Inxa0vitro, GzmB cleaved the endothelial junction protein, vascular endothelial (VE)-cadherin, resulting in the disruption of endothelial barrier function. Together, these results suggest a perforin-independent, extracellular role for GzmB in the pathogenesis of cardiac fibrosis.

CrossTalk proposal: The late sodium current is an important player in the development of diastolic heart failure (heart failure with a preserved ejection fraction)

Approximately half of all patients with heart failure have an ejection fraction greater than 40–50% and may be diagnosed as having Heart Failure with preserved Ejection Fraction (HFpEF). Diastolic dysfunction is central to the pathophysiology of HFpEF (Borlaug & Paulus, 2011), and describes the slowing of ventricular relaxation and increased diastolic stiffness which ultimately impairs ventricular filling. The mechanistic basis of this impairment is complex and not yet well understood. Structural remodelling undoubtedly plays an important role in increasing left ventricular stiffness. However, the acute worsening of diastolic dysfunction during stress or exercise characteristic of HFpEF suggests an important contribution from dynamic changes in left ventricular (LV) functional properties. Frequency-dependent elevation of diastolic tension and intracellular Ca2+ ([Ca2+]i) has been observed in cardiac muscle strips from patients with left ventricular hypertrophy and diastolic dysfunction, or heart failure (Sossalla et al. 2008; Selby et al. 2011), implying that dysregulation of [Ca2+]i homeostasis of the cardiomyocyte contributes to diastolic dysfunction. n nIntracellular Ca2+ regulation is closely linked to intracellular Na+ homeostasis, through the Na+–Ca2+ exchanger (NCX). Intracellular Na+ of cardiomyocytes from failing hearts is increased and associated with elevated diastolic tension (Pieske et al. 2002). An important mechanism underlying this observation may be an increase in the late sodium current (INa,L). The Na+ conductance responsible for rapid depolarization of cardiomyocytes does not completely inactivate during the action potential. (Noble & Noble, 2006; Maier, 2012) Some channels continue to conduct, or even reactivate at relatively positive membrane potentials during the plateau and repolarization phases. This is INa,L (Zaza et al. 2008). Consequently, about half of the myocyte Na+ entry occurs during the initial 2–3xa0ms, and about half during the remainder of the action potential (Makielski & Farley, 2006). At the molecular level, INa,L results from channel reopening during sustained depolarization by two different modes of gating: burst openings and late scattered openings (Maltsev & Undrovinas, 2008). n nAs outlined in Fig.xa01, increased Na+ entry through INa,L increases intracellular Na+ ([Na+]i), which reduces the driving force for extrusion of Ca2+ and favours Ca2+ influx via the Na+–Ca2+ exchanger (NCX). This leads to increased [Ca2+]i. Elevated [Ca2+]i eventually increases actin–myosin filament interaction during diastole and thus increases diastolic tension. This mechanism of Ca2+ overload has been demonstrated in numerous animal studies, and in strips of ventricular muscle or myocytes isolated from patients with failing hearts (Valdivia et al. 2005; Makielski & Farley, 2006; Maltsev & Undrovinas, 2008; Sossalla et al. 2008; Selby et al. 2011; Coppini et al. 2013). Further, specific augmentation of INa,L with the sea anemone toxin ATXII in isolated myocytes and perfused hearts results in Na+ and Ca2+ overload (Fraser et al. 2006; Sossalla et al. 2008) and impaired diastolic function. Diastolic dysfunction with preserved systolic function has also been described in LQT syndrome type 3 patients, where INa,L is enhanced due to a Na+ channel mutation (Moss et al. 2008; Hummel et al. 2013). n n n nFigure 1 n nA pathological enhanced INa,L contributes to Na+-dependent Ca2+ overload, diastolic dysfunction n n n nWe propose that a pathological increase in Na+ influx through cardiac Na+ channels, specifically due to enhanced INa,L is a major contributor to Ca2+ overload and diastolic dysfunction in HFpEF. Key evidence to support this hypothesis is outlined below.

Rebuttal from Marc Pourrier, Sarah Williams, Donald McAfee, Luiz Belardinelli and David Fedida

Papp et al. (2013) raise a number of salient points in disputing the importance of INa,L in diastolic dysfunction in HFpEF. First, they question the selective effect of enhanced INa,L on passive relaxation when one would expect increased INa,L to affect early active relaxation kinetics as well. While the passive phase of relaxation is dependent on diastolic Ca2+ accumulation and Ca2+ extrusion primarily via the Na+–Ca2+ exchanger, the active phase of relaxation, on the other hand is more related to the rate of Ca2+ reuptake into the sarcoplasmic reticulum (SR). Although specific enhancement of INa,L in cardiac myocytes using the sea anemone toxin ATXII slows Ca2+ reuptake into the SR (Sossalla et al. 2008), experimental and clinical data obtained with ranolazine indicate it has mixed effects on active relaxation (Moss et al. 2008; Sossalla et al. 2008; Undrovinas et al. 2010; Coppini et al. 2013; Maier et al. 2013). These discrepancies suggest that the contribution of INa,L to active relaxation may differ in various diseases. n nPapp et al. also argue that the role of CaMKII in increased INa,L-induced Na+-dependent Ca2+ accumulation in human cardiac myocytes has yet to be demonstrated to be directly associated with diastolic dysfunction and HFpEF. While experiments in human failing myocytes are lacking, there is experimental evidence that associates CaMKII with INa,L, Na+-dependent Ca2+ accumulation and diastolic dysfunction. Specifically, (1) activity of CaMKII is increased in human heart failure (HF) and diastolic dysfunction (Sossalla et al. 2010; Coppini et al. 2013) and (2) CaMKII phosphorylates Na+ channels, resulting in enhanced INa,L, and elevation of intracellular Na+ (Wagner et al. 2006). It is therefore reasonable to consider that CaMKII, as well as other modulators play an important role in INa,L-induced Na+-dependent Ca2+ accumulation and diastolic dysfunction in HFpEF (Zaza et al. 2008). n nRanolazine is considered the prototypical INa,L inhibitor, and we agree with Papp et al. that it has other actions able to account for some of the effects observed on diastolic function. However, it is unlikely that the effects of ranolazine on myocardial relaxation are related to pFOX inhibition, as high concentrations (12% inhibition at 100xa0μm) are required, whereas cardiac function is improved in the presence of ∼10xa0μm ranolazine. n nMore compelling is the fact that the specific Na+ channel inhibitor tetrodotoxin attenuates INa,L-induced Na+-dependent Ca2+ accumulation in failing ventricular myocytes (Undrovinas et al. 2010). Similarly, the novel highly specific and selective INa,L inhibitor GS967 decreases ATXII-induced increase in intracellular Na+ and diastolic Ca2+ in isolated rabbit ventricular myocytes (Belardinelli et al. 2013).