Loss of ADAM15 in female mice does not worsen pressure overload cardiomyopathy, independent of ovarian hormones.

Cardiac hypertrophy is a common feature in several cardiomyopathies. We previously reported that loss of ADAM15 (disintegrin and metalloproteinase 15) worsened cardiac hypertrophy and dilated cardiomyopathy following cardiac pressure overload. Here, we investigated the impact of ADAM15 loss in female mice following cardiac pressure overload induced by transverse aortic constriction (TAC). Female Adam15-/-mice developed the same degree of cardiac hypertrophy, dilation and dysfunction as the parallel female wildtype (WT) mice at 6 weeks post-TAC. To determine if this is due to the protective effects of estrogen which could mask the negative impact of Adam15 loss, WT and Adam15-/- mice underwent ovariectomy (OVx) 2 weeks prior to TAC. Cardiac structure and function analyses were performed at 6 weeks post-TAC. OVx similarly impacted females of both genotypes post-TAC. Calcineurin (Cn) activity was increased post-OVx-TAC, and more in Adam15-/- mice, however this increase was not reflected in the total-to-phospho NFAT levels. Integrin α7 expression, which was upstream of Cn activation in male Adam15-/--TAC mice, remained unchanged in female mice. However, activation of the Mitogen Activated Protein Kinases (ERK, JNK, P38) were greater in Adam15-/--OVx-TAC compared to WT-OVx-TAC mice. In addition, ADAM15 protein levels were significantly increased post-TAC in male but not in female WT mice. Myocardial fibrosis was comparable in non-OVx WT-TAC and Adam15-/--TAC mice. OVx increased the perivascular fibrosis more in Adam15-/- compared to WT mice post-TAC. Our data demonstrate that loss of ovarian hormones did not fully replicate the male phenotype in the female Adam15-/- mice post-TAC. Since ADAM15 levels were increased in males but not in females post-TAC, it is plausible that ADAM15 does not play a prominent role in post-TAC events in female mice. Our findings highlight the significance of factors other than sex hormones in mediating cardiomyopathies in females, which require a more thorough understanding.

Loss of TIMP3, but not TIMP4, exacerbates thoracic and abdominal aortic aneurysm.

AIMS: Aorta exhibits regional heterogeneity (structural and functional), while different etiologies for thoracic and abdominal aortic aneurysm (TAA, AAA) are recognized. Tissue inhibitor of metalloproteinases (TIMPs) regulate vascular remodeling through different mechanisms. Region-dependent functions have been reported for TIMP3 and TIMP4 in vascular pathologies. We investigated the region-specific function of these TIMPs in development of TAA versus AAA. METHODS & RESULTS: TAA or AAA was induced in male and female mice lacking TIMP3 (Timp3-/-), TIMP4 (Timp4-/-) or in wildtype (WT) mice by peri-adventitial elastase application. Loss of TIMP3 exacerbated TAA and AAA severity in males and females, with a greater increase in proteinase activity, smooth muscle cell phenotypic switching post-AAA and -TAA, while increased inflammation was detected in the media post-AAA, but in the adventitia post-TAA. Timp3-/- mice showed impaired intimal barrier integrity post-AAA, but a greater adventitial vasa-vasorum branching post-TAA, which could explain the site of inflammation in AAA versus TAA. Severity of TAA and AAA in Timp4-/- mice was similar to WT mice. In vitro, Timp3 knockdown more severely compromised the permeability of human aortic EC monolayer compared to Timp4 knockdown or the control group. In aneurysmal aorta specimens from patients, TIMP3 expression decreased in the media in AAA, and in adventitial in TAA specimens, consistent with the impact of its loss in AAA versus TAA in mice. CONCLUSION: TIMP3 loss exacerbates inflammation, adverse remodeling and aortic dilation, but triggers different patterns of remodeling in AAA versus TAA, and through different mechanisms.

Endothelial phosphoinositide 3-kinase-ß inactivation confers protection from immune-mediated vascular injury.

Heart transplant and recipient survival are limited by immune cell-mediated injury of the graft vasculature. We examined the role of the phosphoinositide 3-kinase-ß (PI3Kß) isoform in endothelial cells (EC) during coronary vascular immune injury and repair in mice. In minor histocompatibility-antigen mismatched allogeneic heart grafts, a robust immune response was mounted to each wild-type, PI3Kß inhibitor-treated, or endothelial-selective PI3Kß knockout (ECßKO) graft transplanted to wild-type recipients. However, microvascular EC loss and progressive occlusive vasculopathy only developed in control, but not PI3Kß-inactivated hearts. We observed a delay in inflammatory cell infiltration of the ECßKO grafts, particularly in the coronary arteries. Surprisingly, this was accompanied by an impaired display of proinflammatory chemokine and adhesion molecules by the ECßKO ECs. In vitro, tumor necrosis factor α-stimulated endothelial ICAM1 and VCAM1 expression was blocked by PI3Kß inhibition or RNA interference. Selective PI3Kß inhibition also blocked tumor necrosis factor α-stimulated degradation of inhibitor of nuclear factor kappa Bα and nuclear translocation of nuclear factor kappa B p65 in EC. These data identify PI3Kß as a therapeutic target to reduce vascular inflammation and injury.

Loss of ADAM15 Exacerbates Transition to Decompensated Myocardial Hypertrophy and Dilation Through Activation of the Calcineurin Pathway.

BACKGROUND: Myocardial hypertrophy and dilation are key features of cardiomyopathies and involve several cellular and molecular events. ADAMs (a disintegrin and metalloproteinases) are membrane-bound proteinases with diverse functions whose role in heart disease remains underexplored. ADAM15 is expressed in the heart and is downregulated in the failing human heart. We investigated the role ADAM15 in pressure overload cardiomyopathy. METHODS: We assessed ADAM15 levels in myocardial specimens from patients. Its direct role in pressure overload was investigated by subjecting wildtype and Adam15-deficient mice to transverse aortic constriction (TAC). RESULTS: ADAM15 levels did not change in patients with concentric hypertrophy, but markedly decreased in eccentric hypertrophy and heart failure. Loss of ADAM15 alone did not cause cardiomyopathy in mice (1 year old). After TAC, Adam15-/- mice exhibited worsened eccentric hypertrophy and dilation with greater increase in hypertrophy markers (pJNK, pERK1/2; Nppb, Nppa, Myh7, Acta1) compared with wildtype-TAC. Expression of integrin-α7 (but not integrin ß1) increased significantly more in Adam15-/--TAC hearts, while the interaction of these integrins with basement membrane (laminin), decreased consistent with worsened left ventricle dilation. In vitro, ADAM15 knockdown increased cardiomyocyte hypertrophy in response to mechanical stretch. Adam15-/--TAC hearts exhibited increased calcineurin activity and de-phosphorylation of nuclear factor of activated T cells. Calcineurin inhibition (cyclosporin-A) blocked the excess hypertrophy and dilation in Adam15-/--TAC mice. Proteome profiling demonstrated the increased abundance of the key proteins linked to worsened DCM in Adam15-/--TAC. CONCLUSION: This is the first report demonstrating that ADAM15 can suppress hypertrophy through regulating the integrin-laminin interaction and the calcineurin pathway.

High-impact opportunities to address ischemia: a focus on heart and circulatory research.

Myocardial ischemic injury and its resolution are the key determinants of morbidity or mortality in heart failure. The cause and duration of ischemia in patients vary. Numerous experimental models and methods have been developed to define genetic, metabolic, molecular, cellular, and pathophysiological mechanisms, in addition to defining structural and functional deterioration of cardiovascular performance. The rapid rise of big data, such as single-cell analysis techniques with bioinformatics, machine learning, and neural networking, brings a new level of sophistication to our understanding of myocardial ischemia. This mini-review explores the multifaceted nature of ischemic injury in the myocardium. We highlight recent state-of-the-art findings and strategies to show new directions of high-impact approach to understanding myocardial tissue remodeling. This next age of heart and circulatory physiology research will be more comprehensive and collaborative to uncover the origin, progression, and manifestation of heart failure while strengthening novel treatment strategies.

SARS-CoV-2 infection downregulates myocardial ACE2 and potentiates cardiac inflammation in humans and hamsters.

Myocardial pathologies resulting from SARS-CoV-2 infections are consistently rising with mounting case rates and reinfections; however, the precise global burden is largely unknown and will have an unprecedented impact. Understanding the mechanisms of COVID-19-mediated cardiac injury is essential toward the development of cardioprotective agents that are urgently needed. Assessing novel therapeutic strategies to tackle COVID-19 necessitates an animal model that recapitulates human disease. Here, we sought to compare SARS-CoV-2-infected animals with patients with COVID-19 to identify common mechanisms of cardiac injury. Two-month-old hamsters were infected with either the ancestral (D614) or Delta variant (B.1.617.2) of SARS-CoV-2 for 2 days, 7 days, and/or 14 days. We measured viral RNA and cytokine expression at the earlier time points to capture the initial stages of infection in the lung and heart. We assessed myocardial angiotensin-converting enzyme 2 (ACE2), the entry receptor for the SARS-CoV-2 virus, and cardioprotective enzyme, as well as markers for inflammatory cell infiltration in the hamster hearts at days 7 and 14. In parallel, human hearts were stained for ACE2, viral nucleocapsid, and inflammatory cells. Indeed, we identify myocardial ACE2 downregulation and myeloid cell burden as common events in both hamsters and humans infected with SARS-CoV-2, and we propose targeting downstream ACE2 downregulation as a therapeutic avenue that warrants clinical investigation.NEW & NOTEWORTHY Cardiac manifestations of COVID-19 in humans are mirrored in the SARS-CoV-2 hamster model, recapitulating myocardial damage, ACE2 downregulation, and a consistent pattern of immune cell infiltration independent of viral dose and variant. Therefore, the hamster model is a valid approach to study therapeutic strategies for COVID-19-related heart disease.

Scleraxis and fibrosis in the pressure-overloaded heart.

AIMS: In response to pro-fibrotic signals, scleraxis regulates cardiac fibroblast activation in vitro via transcriptional control of key fibrosis genes such as collagen and fibronectin; however, its role in vivo is unknown. The present study assessed the impact of scleraxis loss on fibroblast activation, cardiac fibrosis, and dysfunction in pressure overload-induced heart failure. METHODS AND RESULTS: Scleraxis expression was upregulated in the hearts of non-ischemic dilated cardiomyopathy patients, and in mice subjected to pressure overload by transverse aortic constriction (TAC). Tamoxifen-inducible fibroblast-specific scleraxis knockout (Scx-fKO) completely attenuated cardiac fibrosis, and significantly improved cardiac systolic function and ventricular remodelling, following TAC compared to Scx+/+ TAC mice, concomitant with attenuation of fibroblast activation. Scleraxis deletion, after the establishment of cardiac fibrosis, attenuated the further functional decline observed in Scx+/+ mice, with a reduction in cardiac myofibroblasts. Notably, scleraxis knockout reduced pressure overload-induced mortality from 33% to zero, without affecting the degree of cardiac hypertrophy. Scleraxis directly regulated transcription of the myofibroblast marker periostin, and cardiac fibroblasts lacking scleraxis failed to upregulate periostin synthesis and secretion in response to pro-fibrotic transforming growth factor ß. CONCLUSION: Scleraxis governs fibroblast activation in pressure overload-induced heart failure, and scleraxis knockout attenuated fibrosis and improved cardiac function and survival. These findings identify scleraxis as a viable target for the development of novel anti-fibrotic treatments.

Implications of Sm22α-Cre expression in keratinocytes and unanticipated inflammatory skin lesion in a model of atherosclerosis.

Genetically modified mice are widely used to recapitulate human diseases. Atherosclerosis can be induced in mice with low-density lipoprotein receptor (Ldlr)-deficiency and a high-fat diet (HFD). Disintegrin and metalloproteinase-17 (ADAM17) in the smooth muscle cell (SMC) contribute to vascular pathologies, and hence its role in atherosclerosis was investigated. Adam17 deletion in SMCs by Sm22α-Cre driver (Ldlr-/-/Adam17Sm22Cre) and HFD resulted in severe skin lesions in >70% of mice, associated with skin inflammation, which was not observed in Ldlr-/--HFD, nor in mice with SMC deficiency of Adam17 by a different Cre driver (Ldlr-/-/Adam17Myh11Cre). We found that Sm22α is highly expressed in keratinocytes (compared with SMCs), which could underlie the observed skin lesion in Ldlr-/-/Adam17Sm22Cre-HFD. Although expression of Sm22α in non-SMCs has been reported, this is the first study demonstrating a severe side effect resulting from the off-target expression of Sm22α-Cre, resulting in ADAM17 loss in keratinocytes that led to a moribund state.NEW & NOTEWORTHY Although Sm22α-Cre is commonly used to target gene deletion in smooth muscle cells, Sm22α-derived Adam17 deletion resulted in unexpected severe skin lesions following high-fat diet feeding in a model of atherosclerosis. Adam17 deletion by a different SMC driver, Myh11-Cre, did not result in skin lesions in the same atherosclerosis model. Sm22α is highly expressed in keratinocytes, causing ectopic loss of ADAM17 in keratinocytes that caused significant epidermal lesions when combined with a high-fat diet.

Cardiomyocyte-specific disruption of soluble epoxide hydrolase limits inflammation to preserve cardiac function.

Endotoxemia elicits a multiorgan inflammatory response that results in cardiac dysfunction and often leads to death. Inflammation-induced metabolism of endogenous N-3 and N-6 polyunsaturated fatty acids generates numerous lipid mediators, such as epoxy fatty acids (EpFAs), which protect the heart. However, EpFAs are hydrolyzed by soluble epoxide hydrolase (sEH), which attenuates their cardioprotective actions. Global genetic disruption of sEH preserves EpFA levels and attenuates cardiac dysfunction in mice following acute lipopolysaccharide (LPS)-induced inflammatory injury. In leukocytes, EpFAs modulate the innate immune system through the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. However, the mechanisms by which both EpFAs and sEH inhibition exert their protective effects in the cardiomyocyte are still elusive. This study investigated whether cardiomyocyte-specific sEH disruption attenuates inflammation and cardiac dysfunction in acute LPS inflammatory injury via modulation of the NLRP3 inflammasome. We use tamoxifen-inducible CreER recombinase technology to target sEH genetic disruption to the cardiomyocyte. Primary cardiomyocyte studies provide mechanistic insight into inflammasome signaling. For the first time, we demonstrate that cardiomyocyte-specific sEH disruption preserves cardiac function and attenuates inflammatory responses by limiting local cardiac inflammation and activation of the systemic immune response. Mechanistically, inhibition of cardiomyocyte-specific sEH activity or exogenous EpFA treatment do not prevent upregulation of NLRP3 inflammasome machinery in neonatal rat cardiomyocytes. Rather, they limit downstream activation of the pathway leading to release of fewer chemoattractant factors and recruitment of immune cells to the heart. These data emphasize that cardiomyocyte sEH is vital for mediating detrimental systemic inflammation.NEW & NOTEWORTHY The cardioprotective effects of genetic disruption and pharmacological inhibition of sEH have been demonstrated in a variety of cardiac disease models, including acute LPS inflammatory injury. For the first time, it has been demonstrated that sEH genetic disruption limited to the cardiomyocyte profoundly preserves cardiac function and limits local and systemic inflammation following acute LPS exposure. Hence, cardiomyocytes serve a critical role in the innate immune response that can be modulated to protect the heart.

Disintegrin and Metalloproteinases (ADAMs [A Disintegrin and Metalloproteinase] and ADAMTSs [ADAMs With a Thrombospondin Motif]) in Aortic Aneurysm.

Aortic aneurysm is a complex pathology that can be lethal if not detected in time. Although several molecular mechanisms and pathways have been identified to be involved in aortic aneurysm development and growth, the current lack of an effective pharmacological treatment highlights the need for a more thorough understanding of the factors that regulate the remodeling of the aortic wall in response to triggers that lead to aneurysm formation. This task is further complicated by the regional heterogeneity of the aorta and that thoracic and abdominal aortic aneurysm are distinct pathologies with different risk factors and distinct course of progression. ADAMs (a disintegrin and metalloproteinases) and ADAMTS (ADAMs with a thrombospondin motif) are proteinases that share similarities with other proteinases but possess unique and diverse properties that place them in a category of their own. In this review, we discuss what is known on how ADAMs and ADAMTSs are altered in abdominal aortic aneurysm and thoracic aortic aneurysm in patients, in different animal models, and their role in regulating the function of different vascular and inflammatory cell types. A full understanding of the role of ADAMs and ADAMTSs in aortic aneurysm will help reveal a more complete understanding of the underlying mechanism driving aneurysm formation, which will help towards developing an effective treatment in preventing or limiting the growth of aortic aneurysm.

ADAM15 is required for optimal collagen cross-linking and scar formation following myocardial infarction.

Collagen cross-linking is an important step in optimal scar formation. Myocardial infarction (MI) results in loss of cardiomyocytes that are replaced with a scar (infarct) tissue. Disintegrin and metalloproteinases (ADAMs) are membrane-bound proteases that can interact with molecules intra- and extra-cellularly to mediate various cellular functions. ADAM15 is expressed in the myocardium, however its function in heart disease has been poorly explored. We utilized mice lacking ADAM15 (Adam15-/-) and wildtype (WT) mice. MI, induced by ligation of the left anterior descending artery, resulted in a transient but significant rise in ADAM15 protein in the WT myocardium at 3-days. Following MI, Adam15-/- mice exhibited markedly higher rate of left ventricular (LV) rupture compared to WT mice (66% vs. 15%, p<0.05). Echocardiography and strain analyses showed worsened LV dysfunction in Adam15-/- mice at 3days, prior to the onset of LV rupture. Second harmonic generation imaging revealed significant disarray and reduction in fibrillar collagen density in Adam15-/- compared to WT hearts. This was associated with lower insoluble and higher soluble collagen fractions, reduced cross-linking enzyme, lysyl oxidase-1 (LOX-1), and fibronectin which is required for LOX-1 function, in Adam15-/--MI hearts. Post-MI myocardial inflammation was comparable between the genotypes. In vitro, primary adult cardiac fibroblasts from Adam15-/- mice showed suppressed activation in response to ischemia (hypoxia+nutrient depletion) compared to WT fibroblasts. Adam15-deficiency was associated with reduced PAK1(p21-activated kinase-1) levels, a regulator of fibronectin and LOX-1 expression. In female mice, the rate of post-MI LV rupture, PAK1 signaling, LOX-1 and fibronectin protein levels were comparable between Adam15-/- and WT, indicating less impact of ADAM15 loss in females post- MI. This study reports a novel function for ADAM15 in collagen cross-linking and optimal scar formation post-MI which may also apply to scar formation in other tissues.

Dysregulation of ACE (Angiotensin-Converting Enzyme)-2 and Renin-Angiotensin Peptides in SARS-CoV-2 Mediated Mortality and End-Organ Injuries.

ACE (angiotensin-converting enzyme)-2 as the target for SARS-CoV-2 also negatively regulates the renin-angiotensin system. Pathological activation of ADAM17 (A disintegrin and metalloproteinase-17) may potentiate inflammation and diminish ACE2-mediated tissue protection through proteolytic shedding, contributing to SARS-CoV-2 pathogenesis. We aim to examine plasma soluble ACE2 and angiotensin profiles in relation to outcomes by enrolling consecutive patients admitted for COVID-19 with baseline blood collection at admission and repeated sampling at 7 days. The primary outcome was 90-day mortality, and secondary outcomes were the incidence of end-organ injuries. Overall, 242 patients were included, the median age was 63 (52-74) years, 155 (64.0%) were men, and 57 (23.6%) patients reached the primary end point. Baseline soluble ACE2 was elevated in COVID-19 but was not associated with disease severity or mortality. In contrast, an upward trajectory of soluble ACE2 at repeat sampling was independently associated with an elevated risk of mortality and incidence of acute myocardial injury and circulatory shock. Similarly, an increase in soluble tumor necrosis factor receptor levels was also associated with adverse outcomes. Plasma Ang I, Ang 1-7 (angiotensin 1-7) levels, and the Ang 1-7/Ang II (angiotensin II) ratio were elevated during SARS-CoV-2 infection related to downregulation of ACE activity at baseline. Moreover, patients having an upward trajectory of soluble ACE2 were characterized by an imbalance in the Ang 1-7/Ang II ratio. The observed dysregulation of ACE2 and angiotensin peptides with disease progression suggest a potential role of ADAM17 inhibition and enhancing the beneficial Ang 1-7/Mas axis to improve outcomes against SARS-CoV-2 infection.