Structural insights into regulation of CCN protein activities and functions.

CCN proteins play important functions during development, in repair mechanisms following tissue injury, as well as in pathophysiologic mechanisms of metastasis of cancer. CCNs are secreted proteins that have a multimodular structure and are categorized as matricellular proteins. Although the prevailing view is that CCN proteins regulate biologic processes by interacting with a wide array of other proteins in the microenvironment of the extracellular matrix, the molecular mechanisms of action of CCN proteins are still poorly understood. Not dissuading the current view, however, the recent appreciation that these proteins are signaling proteins in their own right and may even be considered preproproteins controlled by endopeptidases to release a C-terminal bioactive peptide has opened new avenues of research. Also, the recent resolution of the crystal structure of two of the domains of CCN3 have provided new knowledge with implications for the entire CCN family. These resolved structures in combination with structural predictions based upon the AlphaFold artificial intelligence tool provide means to shed new light on CCN functions in context of the notable literature in the field. CCN proteins have emerged as important therapeutic targets in several disease conditions, and clinical trials are currently ongoing. Thus, a review that critically discusses structure - function relationship of CCN proteins, in particular as it relates to interactions with other proteins in the extracellular milieu and on the cell surface, as well as to cell signaling activities of these proteins, is very timely. Suggested mechanism for activation and inhibition of signaling by the CCN protein family (graphics generated with BioRender.com ).

Report on the 11th international workshop on the CCN family of genes, Nice, October 20-24, 2022.

In celebration of the twentieth anniversary of the inception of the CCN society, and of the first post-Covid-19 live meeting, the executive board of the ICCNS had chosen Nice as the venue for the 11th International workshop on the CCN family of genes. On this occasion participation in the meeting was extended to colleagues from other cell signaling fields who were invited to present both an overview of their work and the future directions of their laboratory. Also, for the first time, the members of the JCCS Editorial Board were invited to participate in a JCCS special session during which all aspects of the journal « life ¼ were addressed and opened to free critical discussion. The scientific presentations and the discussions that followed showed once more that an expansion of the session topics was beneficial to the quality of the meeting and confirmed that the ARBIOCOM project discussed last April in Nice was now on track to be launched in 2023. The participants unanimously welcomed Professor Attramadal's proposition to organize the 2024, 12th International CCN workshop in Oslo, Norway.

The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2.

Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, that is, induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF-7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.

Cardiomyocyte-specific overexpression of syndecan-4 in mice results in activation of calcineurin-NFAT signalling and exacerbated cardiac hypertrophy.

BACKGROUND: Cardiomyocyte hypertrophy is a hallmark of cardiac dysfunction in patients with aortic stenosis (AS), and can be triggered by left ventricular (LV) pressure overload in mice by aortic banding (AB). Syndecan-4 is a transmembrane heparan sulphate proteoglycan which is found increased in the myocardium of AS patients and AB mice. The role of syndecan-4 in cardiomyocyte hypertrophy is not well understood. PURPOSE OF THE STUDY: We developed mice with cardiomyocyte-specific overexpression of syndecan-4 (Sdc4-Tg) and subjected these to AB to examine the role of syndecan-4 in hypertrophy and activation of the pro-hypertrophic calcineurin-NFAT signalling pathway. METHODS AND RESULTS: Sdc4-Tg mice showed exacerbated cardiac remodelling upon AB compared to wild type (WT). At 2-6 weeks post-AB, Sdc4-Tg and WT mice showed similar hypertrophic growth, while at 20 weeks post-AB, exacerbated hypertrophy and dysfunction were evident in Sdc4-Tg mice. After cross-breeding of Sdc4-Tg mice with NFAT-luciferase reporter mice, we found increased NFAT activation in Sdc4-Tg hearts after AB. Immunoprecipitation showed that calcineurin bound to syndecan-4 in Sdc4-Tg hearts. Isolated cardiomyocytes from Sdc4-Tg mice showed alterations in Ca2+ fluxes, suggesting that syndecan-4 regulated Ca2+ levels, and thereby, activating the syndecan-4-calcineurin complex resulting in NFAT activation and hypertrophic growth. Similarly, primary cardiomyocyte cultures from neonatal rats showed increased calcineurin-NFAT-dependent hypertrophic growth upon viral Sdc4 overexpression. CONCLUSION: Our study of mice with cardiomyocyte-specific overexpression of Sdc4 have revealed that syndecan-4 is important for activation of the Ca2+-dependent calcineurin-NFAT signalling pathway, hypertrophic remodelling and dysfunction in cardiomyocytes in response to pressure overload.

Impact of delayed type hypersensitivity arthritis on development of heart failure by aortic constriction in mice.

AIMS: Patients with rheumatoid arthritis (RA) have increased risk of heart failure (HF). The mechanisms and cardiac prerequisites explaining this association remain unresolved. In this study, we sought to determine the potential cardiac impact of an experimental model of RA in mice subjected to HF by constriction of the ascending aorta. METHODS: Aorta was constricted via thoracotomy and placement of o-rings with inner diameter 0.55 mm or 0.66 mm, or sham operated. RA-like phenotype was instigated by delayed-type hypersensitivity arthritis (DTHA) two weeks after surgery and re-iterated after additional 18 days. Cardiac magnetic resonance imaging (MRI) was performed before surgery and at successive time points throughout the study. Six weeks after surgery the mice were euthanized, blood and tissue were collected, organ weights were documented, and expression levels of cardiac foetal genes were analysed. In a supplemental study, DTHA-mice were euthanized throughout 14 days after induction of arthritis, and blood was analysed for important markers and mediators of RA (SAP, TNF-α and IL-6). In order to put the latter findings into clinical context, the same molecules were analysed in serum from untreated RA patients and compared to healthy controls. RESULTS: Significant elevations of inflammatory markers were found in both patient- and murine blood. Furthermore, the DTHA model appeared clinically relevant when compared to the inflammatory responses observed in three prespecified RA severity disease states. Two distinct trajectories of cardiac dysfunction and HF development were found using the two o-ring sizes. These differences were consistent by both MRI, organ weights and cardiac foetal gene expression levels. Still, no difference within the HF groups, nor within the sham groups, could be found when DTHA was induced. CONCLUSION: DTHA mediated systemic inflammation did not cause, nor modify HF caused by aortic constriction. This indicates other prerequisites for RA-induced cardiac dysfunction.

Tissue distribution and transcriptional regulation of CCN5 in the heart after myocardial infarction.

CCN5 is a divergent member of the cellular communication network factor (CCN) family in that it lacks the carboxyl terminal cystine knot domain common to the other CCN family members. CCN5 has been reported to antagonize the profibrotic actions of CCN2 and to inhibit myocardial collagen deposition and fibrosis in chronic pressure overload of the heart. However, what mechanisms that regulate CCN5 activity in the heart remain unknown. Recombinant, replication defective adenovirus encoding firefly luciferase under control of the human CCN5 promoter was prepared and used to investigate what mechanisms regulate CCN5 transcription in relevant cells. Tissue distribution of CCN5 in hearts from healthy mice and from mice subjected to myocardial infarction was investigated. Contrary to the profibrotic immediate early gene CCN2, we find that CCN5 is induced in the late proliferation and maturation phases of scar healing. CCN5 was identified principally in endothelial cells, fibroblasts, smooth muscle cells, and macrophages. Our data show that CCN5 gene transcription and protein levels are induced by catecholamines via ß2-adrenergic receptors. Myocardial induction of CCN5 was further confirmed in isoproterenol-infused mice. We also find that CCN5 transcription is repressed by TNF-α, an inflammatory mediator highly elevated in early phases of wound healing following myocardial infarction. In conclusion, CCN5 predominates in endothelial cells, fibroblasts, and macrophages of the differentiating scar tissue and its transcription is conversely regulated by ß2-adrenergic agonists and TNF-α.

Instability in NAD+ metabolism leads to impaired cardiac mitochondrial function and communication.

Poly(ADP-ribose) polymerase (PARP) enzymes initiate (mt)DNA repair mechanisms and use nicotinamide adenine dinucleotide (NAD+) as energy source. Prolonged PARP activity can drain cellular NAD+ reserves, leading to de-regulation of important molecular processes. Here, we provide evidence of a pathophysiological mechanism that connects mtDNA damage to cardiac dysfunction via reduced NAD+ levels and loss of mitochondrial function and communication. Using a transgenic model, we demonstrate that high levels of mice cardiomyocyte mtDNA damage cause a reduction in NAD+ levels due to extreme DNA repair activity, causing impaired activation of NAD+-dependent SIRT3. In addition, we show that myocardial mtDNA damage in combination with high dosages of nicotinamideriboside (NR) causes an inhibition of sirtuin activity due to accumulation of nicotinamide (NAM), in addition to irregular cardiac mitochondrial morphology. Consequently, high doses of NR should be used with caution, especially when cardiomyopathic symptoms are caused by mitochondrial dysfunction and instability of mtDNA.

Exercise training reveals micro-RNAs associated with improved cardiac function and electrophysiology in rats with heart failure after myocardial infarction.

AIMS: Endurance training improves aerobic fitness and cardiac function in individuals with heart failure. However, the underlying mechanisms are not well characterized. Exercise training could therefore act as a tool to discover novel targets for heart failure treatment. We aimed to associate changes in Ca2+ handling and electrophysiology with micro-RNA (miRNA) profile in exercise trained heart failure rats to establish which miRNAs induce heart failure-like effects in Ca2+ handling and electrophysiology. METHODS AND RESULTS: Post-myocardial infarction (MI) heart failure was induced in Sprague Dawley rats. Rats with MI were randomized to sedentary control (sed), moderate (mod)- or high-intensity (high) endurance training for 8 weeks. Exercise training improved cardiac function, Ca2+ handling and electrophysiology including reduced susceptibility to arrhythmia in an exercise intensity-dependent manner where high intensity gave a larger effect. Fifty-five miRNAs were significantly regulated (up or down) in MI-sed, of which 18 and 3 were changed towards Sham-sed in MI-high and MI-mod, respectively. Thereafter we experimentally altered expression of these "exercise-miRNAs" individually in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM) in the same direction as they were changed in MI. Of the "exercise-miRNAs", miR-214-3p prolonged AP duration, whereas miR-140 and miR-208a shortened AP duration. miR-497-5p prolonged Ca2+ release whereas miR-214-3p and miR-31a-5p prolonged Ca2+ decay. CONCLUSION: Using exercise training as a tool, we discovered that miR-214-3p, miR-497-5p, miR-31a-5p contribute to heart-failure like behaviour in Ca2+ handling and electrophysiology and could be potential treatment targets.

Complement component C3 and the TLR co-receptor CD14 are not involved in angiotensin II induced cardiac remodelling.

Inflammation is centrally involved in the development of cardiac hypertrophy and the processes of remodelling. The complement system and Toll-like receptor (TLR) family, two upstream arms of the innate immune system, have previously been reported to be involved in cardiac remodelling. However, the role of complement component 3 (C3), TLR co-receptor CD14 and the synergy between them have not been addressed during pressure overload-induced cardiac remodelling. Here, we examined angiotensin II-induced cardiac hypertrophy and remodelling for 7 days in male C57Bl/6 J mice deficient in C3, CD14, or both (C3CD14), and WT controls. Angiotensin II infusion induced a mild concentric hypertrophic phenotype in WT mice with increased left ventricle weight, wall thicknesses and reduced ventricular internal diameter, associated with increased cardiac fibrosis. However, there were no differences between WT mice and mice deficient for C3, CD14 or C3CD14, as systolic blood pressure, cardiac function and structure and levels of fibrosis were comparable between WT mice and the three other genotypes. C5a did not change in angiotensin II treated mice, whereas Mac2 levels were increased in angiotensin II treated mice, but did not differ between genotypes. The inflammatory IL-6 response was comparable between WT and C3 deficient mice, however, it was decreased in CD14 and C3CD14 deficient mice. We conclude that deficiency in C3, CD14 or C3CD14 had no effect on cardiac remodelling following angiotensin II-induced pressure overload. This suggests that C3 and CD14 are not involved in angiotensin II-induced adverse cardiac remodelling.

Absence of NLRP3 Inflammasome in Hematopoietic Cells Reduces Adverse Remodeling After Experimental Myocardial Infarction.

An inflammatory response is required for tissue healing after a myocardial infarction (MI), but the process must be balanced to prevent maladaptive remodeling. This study shows that improved survival and cardiac function following MI, in mice deficient for the NLRP3 inflammasome, can be recapitulated in wild-type mice receiving bone marrow from Nlrp3 -/- mice. This suggests that NLRP3 activation in hematopoietic cells infiltrating in the myocardium increases mortality and late ventricular remodeling. Our data should encourage performing clinical trials directly targeting NLRP3 inflammasome and their inflammatory cytokines (interleukin-1ß and -18) in MI patients.

Low Cellular NAD+ Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes.

NAD+ is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD+ on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD+ affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD+ was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD+ levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD+ enhanced IL-1ß release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD+ depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD+ depletion, specifically TAK1, IKKß, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD+ affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD+ and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.

Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation.

Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.

Toll-Like Receptor 9 Promotes Survival in SERCA2a KO Heart Failure Mice.

Aim. Inflammation is important in heart failure (HF). The role of the immune receptor toll-like receptor 9 (TLR9) in HF is not understood and not investigated in diastolic HF. We investigated the role of TLR9 in a murine diastolic HF model caused by cardiomyocyte SERCA2a excision. Methods and Results. We crossed SERCA2a KO and TLR9 KO mice to generate four mouse lines. Tamoxifen-induced cardiomyocyte SERCA2a gene excision was carried out in mice, causing diastolic HF. After 7.6 weeks, cardiac functions and dimensions were analyzed by echocardiography and heart tissues were processed. HF mice depleted of TLR9 demonstrated reduced survival compared to SERC2a KO mice, with a median life expectancy of 58 days compared to 63 days. Both HF groups displayed increased left atrium size, lung weight, fetal gene expressions, monocyte/macrophage infiltration, and fibrosis. However, there were no significant differences between the groups. Conclusion. In mice with SERCA2a KO-induced diastolic HF, the absence of TLR9 reduced median life expectancy. The cause remains elusive, as all investigated HF parameters were unaltered. Still, these findings support a salutary role of TLR9 in some subsets of HF conditions and underline the importance for future studies on the mechanisms of TLR9 in diastolic HF.

Connective tissue growth factor and bone morphogenetic protein 2 are induced following myocardial ischemia in mice and humans.

We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies.

Palmitate promotes inflammatory responses and cellular senescence in cardiac fibroblasts.

Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3-/- and ASC-/-mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1ß, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated ß-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.

CTGF/CCN2 Postconditioning Increases Tolerance of Murine Hearts towards Ischemia-Reperfusion Injury.

BACKGROUND AND PURPOSE: Previous studies of ischemia-reperfusion injury (IRI) in hearts from mice with cardiac-restricted overexpression of CCN2 have shown that CCN2 increases tolerance towards IRI. The objectives of this study were to investigate to what extent post-ischemic administration of recombinant human CCN2 (rhCCN2) would limit infarct size and improve functional recovery and what signaling pathways are involved. EXPERIMENTAL APPROACH: Isolated mice hearts were perfused ad modum Langendorff, subjected to no-flow, global ischemia, and subsequently, exposed to mammalian cell derived, full-length (38-40kDa) rhCCN2 (250 nM) or vehicle during the first 15 min of a 60 min reperfusion period. KEY RESULTS: Post-ischemic administration of rhCCN2 resulted in attenuation of infarct size from 58 ± 4% to 34 ± 2% (p < 0.001) which was abrogated by concomitant administration of the PI3 kinase inhibitor LY294002 (45 ± 3% vs. 50 ± 3%, ns). In congruence with reduction of infarct size rhCCN2 also improved recovery of left ventricular developed pressure (p < 0.05). Western blot analyses of extracts of ex vivo-perfused murine hearts also revealed that rhCCN2 evoked concentration-dependent increase of cardiac phospho-GSK3ß (serine-9) contents. CONCLUSIONS AND IMPLICATIONS: We demonstrate that post-ischemic administration of rhCCN2 increases the tolerance of ex vivo-perfused murine hearts to IRI. Mechanistically, this postconditioning effect of rhCCN2 appeared to be mediated by activation of the reperfusion injury salvage kinase pathway as demonstrated by sensitivity to PI3 kinase inhibition and increased CCN2-induced phosphorylation of GSK3ß (Ser-9). Thus, the rationale for testing rhCCN2-mediated post-ischemic conditioning of the heart in more complex models is established.

NLRP3 inflammasome activation during myocardial ischemia reperfusion is cardioprotective.

BACKGROUND: The innate immune receptor NLRP3 recognizes tissue damage and initiates inflammatory processes through formation multiprotein complexes with the adaptor protein ASC and caspase-1, i.e. NLRP3 inflammasomes, which through cleavage of pro-IL-1ß mediates release of bioactive IL-1ß. We hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction (MI) and sought to investigate the mechanisms herein in an experimental MI model in mice. METHODS AND RESULTS: The left coronary artery (LCA) of WT, NLRP3(-/-) and ASC(-/-) mice of both genders was ligated for 30 min followed by 3 or 24 h reperfusion. For pre-conditioning studies, the TLR2 agonist Pam3CSK4 or PBS was injected intraperitoneally 60 min prior to LCA ligation. For mechanistic investigations, blood plasmas and left ventricle tissues were collected, and a hypothesis-driven selection of protein or mRNA targets was investigated. Surprisingly, hearts from NLRP3-deficient mice featured larger infarct size than WT mice (p = 0.0048). In general, there were only modest changes with no significant pattern in myocardial infiltration of neutrophils and macrophages and systemic and myocardial cytokine expression between the three genotypes. Preconditioning with the TLR2 agonist Pam3CSK4 induced Akt phosphorylation and reduced infarct size in WT but not NLRP3 -or ASC -deficient hearts. CONCLUSION: Absence of NLRP3 results in increased myocardial infarct size after in vivo ischemia reperfusion, seemingly due to dysfunction of the cardioprotective RISK pathway. Our data imply that NLRP3 contributes to cardio-protection during I/R and do not support a role for NLRP3 or ASC inhibition in the management of acute MI including revascularization therapy.

Sustained Toll-Like Receptor 9 Activation Promotes Systemic and Cardiac Inflammation, and Aggravates Diastolic Heart Failure in SERCA2a KO Mice.

AIM: Cardiac inflammation is important in the pathogenesis of heart failure. However, the consequence of systemic inflammation on concomitant established heart failure, and in particular diastolic heart failure, is less explored. Here we investigated the impact of systemic inflammation, caused by sustained Toll-like receptor 9 activation, on established diastolic heart failure. METHODS AND RESULTS: Diastolic heart failure was established in 8-10 week old cardiomyocyte specific, inducible SERCA2a knock out (i.e., SERCA2a KO) C57Bl/6J mice. Four weeks after conditional KO, mice were randomized to receive Toll-like receptor 9 agonist (CpG B; 2µg/g body weight) or PBS every third day. After additional four weeks, echocardiography, phase contrast magnetic resonance imaging, histology, flow cytometry, and cardiac RNA analyses were performed. A subgroup was followed, registering morbidity and death. Non-heart failure control groups treated with CpG B or PBS served as controls. Our main findings were: (i) Toll-like receptor 9 activation (CpG B) reduced life expectancy in SERCA2a KO mice compared to PBS treated SERCA2a KO mice. (ii) Diastolic function was lower in SERCA2a KO mice with Toll-like receptor 9 activation. (iii) Toll-like receptor 9 stimulated SERCA2a KO mice also had increased cardiac and systemic inflammation. CONCLUSION: Sustained activation of Toll-like receptor 9 causes cardiac and systemic inflammation, and deterioration of SERCA2a depletion-mediated diastolic heart failure.

Impaired dynamics and function of mitochondria caused by mtDNA toxicity leads to heart failure.

Cardiac mitochondrial dysfunction has been implicated in heart failure of diverse etiologies. Generalized mitochondrial disease also leads to cardiomyopathy with various clinical manifestations. Impaired mitochondrial homeostasis may over time, such as in the aging heart, lead to cardiac dysfunction. Mitochondrial DNA (mtDNA), close to the electron transport chain and unprotected by histones, may be a primary pathogenetic site, but this is not known. Here, we test the hypothesis that cumulative damage of cardiomyocyte mtDNA leads to cardiomyopathy and heart failure. Transgenic mice with Tet-on inducible, cardiomyocyte-specific expression of a mutant uracil-DNA glycosylase 1 (mutUNG1) were generated. The mutUNG1 is known to remove thymine in addition to uracil from the mitochondrial genome, generating apyrimidinic sites, which obstruct mtDNA function. Following induction of mutUNG1 in cardiac myocytes by administering doxycycline, the mice developed hypertrophic cardiomyopathy, leading to congestive heart failure and premature death after ∼2 mo. The heart showed reduced mtDNA replication, severely diminished mtDNA transcription, and suppressed mitochondrial respiration with increased Pgc-1α, mitochondrial mass, and antioxidative defense enzymes, and finally failing mitochondrial fission/fusion dynamics and deteriorating myocardial contractility as the mechanism of heart failure. The approach provides a model with induced cardiac-restricted mtDNA damage for investigation of mtDNA-based heart disease.