A new approach to characterize cardiac sodium storage by combining fluorescence photometry and magnetic resonance imaging in small animal research.

Cardiac myocyte sodium (Na+) homoeostasis is pivotal in cardiac diseases and heart failure. Intracellular Na+ ([Na+]i) is an important regulator of excitation-contraction coupling and mitochondrial energetics. In addition, extracellular Na+ ([Na+]e) and its water-free storage trigger collagen cross-linking, myocardial stiffening and impaired cardiac function. Therefore, understanding the allocation of tissue Na+ to intra- and extracellular compartments is crucial in comprehending the pathophysiological processes in cardiac diseases. We extrapolated [Na+]e using a three-compartment model, with tissue Na+ concentration (TSC) measured by in vivo 23Na-MRI, extracellular volume (ECV) data calculated from T1 maps, and [Na+]i measured by in vitro fluorescence microscopy using Na+ binding benzofuran isophthalate (SBFI). To investigate dynamic changes in Na+ compartments, we induced pressure overload (TAC) or myocardial infarction (MI) via LAD ligation in mice. Compared to SHAM mice, TSC was similar after TAC but increased after MI. Both TAC and MI showed significantly higher [Na+]i compared to SHAM (around 130% compared to SHAM). Calculated [Na+]e increased after MI, but not after TAC. Increased TSC after TAC was primarily driven by increased [Na+]i, but the increase after MI by elevations in both [Na+]i and [Na+]e.

Mechanistic Insights of the LEMD2 p.L13R Mutation and Its Role in Cardiomyopathy.

BACKGROUND: Nuclear envelope proteins play an important role in the pathogenesis of hereditary cardiomyopathies. Recently, a new form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane protein LEMD2 was discovered. The aim was to unravel the molecular mechanisms of mutant LEMD2 in the pathogenesis of cardiomyopathy. METHODS: We generated a Lemd2 p.L13R knock-in mouse model and a corresponding cell model via CRISPR/Cas9 technology and investigated the cardiac phenotype as well as cellular and subcellular mechanisms of nuclear membrane rupture and repair. RESULTS: Knock-in mice developed a cardiomyopathy with predominantly endocardial fibrosis, left ventricular dilatation, and systolic dysfunction. Electrocardiograms displayed pronounced ventricular arrhythmias and conduction disease. A key finding of knock-in cardiomyocytes on ultrastructural level was a significant increase in nuclear membrane invaginations and decreased nuclear circularity. Furthermore, increased DNA damage and premature senescence were detected as the underlying cause of fibrotic and inflammatory remodeling. As the p.L13R mutation is located in the Lap2/Emerin/Man1 (LEM)-domain, we observed a disrupted interaction between mutant LEMD2 and BAF (barrier-to-autointegration factor), which is required to initiate the nuclear envelope rupture repair process. To mimic increased mechanical stress with subsequent nuclear envelope ruptures, we investigated mutant HeLa-cells upon electrical stimulation and increased stiffness. Here, we demonstrated impaired nuclear envelope rupture repair capacity, subsequent cytoplasmic leakage of the DNA repair factor KU80 along with increased DNA damage, and recruitment of the cGAS (cyclic GMP-AMP synthase) to the nuclear membrane and micronuclei. CONCLUSIONS: We show for the first time that the Lemd2 p.L13R mutation in mice recapitulates human dilated cardiomyopathy with fibrosis and severe ventricular arrhythmias. Impaired nuclear envelope rupture repair capacity resulted in increased DNA damage and activation of the cGAS/STING/IFN pathway, promoting premature senescence. Hence, LEMD2 is a new player inthe disease group of laminopathies.

Dynamics of monocyte-derived macrophage diversity in experimental myocardial infarction.

AIMS: Macrophages have a critical and dual role in post-ischaemic cardiac repair, as they can foster both tissue healing and damage. Multiple subsets of tissue resident and monocyte-derived macrophages coexist in the infarcted heart, but their precise identity, temporal dynamics, and the mechanisms regulating their acquisition of discrete states are not fully understood. To address this, we used multi-modal single-cell immune profiling, combined with targeted cell depletion and macrophage fate mapping, to precisely map monocyte/macrophage transitions after experimental myocardial infarction. METHODS AND RESULTS: We performed single-cell transcriptomic and cell-surface marker profiling of circulating and cardiac immune cells in mice challenged with acute myocardial infarction, and integrated single-cell transcriptomes obtained before and at 1, 3, 5, 7, and 11 days after infarction. Using complementary strategies of CCR2+ monocyte depletion and fate mapping of tissue resident macrophages, we determined the origin of cardiac macrophage populations. The macrophage landscape of the infarcted heart was dominated by monocyte-derived cells comprising two pro-inflammatory populations defined as Isg15hi and MHCII+Il1b+, alongside non-inflammatory Trem2hi cells. Trem2hi macrophages were observed in the ischaemic area, but not in the remote viable myocardium, and comprised two subpopulations sequentially populating the heart defined as Trem2hiSpp1hi monocyte-to-macrophage intermediates, and fully differentiated Trem2hiGdf15hi macrophages. Cardiac Trem2hi macrophages showed similarities to 'lipid-associated macrophages' found in mouse models of metabolic diseases and were observed in the human heart, indicating conserved features of this macrophage state across diseases and species. Ischaemic injury induced a shift of circulating Ly6Chi monocytes towards a Chil3hi state with granulocyte-like features, but the acquisition of the Trem2hi macrophage signature occurred in the ischaemic tissue. In vitro, macrophages acquired features of the Trem2hi signature following apoptotic-cell efferocytosis. CONCLUSION: Our work provides a comprehensive map of monocyte/macrophage transitions in the ischaemic heart, constituting a valuable resource for further investigating how these cells may be harnessed and modulated to promote post-ischaemic heart repair.

Intracellular virus sensor MDA5 mutation develops autoimmune myocarditis and nephritis.

Mutations in IFIH1 gene encoding viral RNA sensor MDA5 have been reported responsible for many interferonopathies, including Aicardi-Goutières syndrome (AGS) and monogenic lupus, however, the pathological link between IFIH1 mutations and various autoimmune symptoms remains unclear. Here, we generated transgenic mice expressing human MDA5 R779H mutant (R779H Tg), reported in AGS and monogenic lupus patient. Mice spontaneously developed myocarditis and nephritis with upregulation of type I IFNs in the major organs. R779H Tg Mavs-/- and R779H Tg Ifnar-/- showed no phenotypes, indicating direct MDA5-signaling pathway involvement. Rag-2 deficiency and bone marrow cells transfer from wild type to adult mice did not prevent myocarditis development, while mice with cardiomyocyte-specific expression of hMDA5 R779H showed cardiomegaly and high expression of inflammatory cytokines. Taken together, our study clarifies that type I IFNs production and chemokines from cardiomyocytes starts in neonatal period and is critical for the development of myocarditis. Activated lymphocytes and auto-antibodies exacerbate the pathogenesis but are dispensable for the onset.

Dynamics of Cardiac Neutrophil Diversity in Murine Myocardial Infarction.

RATIONALE: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage. OBJECTIVE: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction. METHODS AND RESULTS: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G+ (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (Cd177, Lcn2, Fpr1), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb). At 3 and 5 days, 2 major subsets of Siglecfhi (enriched for eg, Icam1 and Tnf) and Siglecflow (Slpi, Ifitm1) neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of Cxcr4, heart infiltrating neutrophils acquired a unique SiglecFhi signature. SiglecFhi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecFhi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecFhi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecFhi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation. CONCLUSIONS: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecFhi signature.

SPRED2 deficiency elicits cardiac arrhythmias and premature death via impaired autophagy.

Cardiac functionality is dependent on a balanced protein turnover. Accordingly, regulated protein decay is critical to maintain cardiac function. Here we demonstrate that deficiency of SPRED2, an intracellular repressor of ERK-MAPK signaling markedly expressed in human heart, resulted in impaired autophagy, heart failure, and shortened lifespan. SPRED2-/- mice showed cardiomyocyte hypertrophy, cardiac fibrosis, impaired electrical excitability, and severe arrhythmias. Mechanistically, cardiomyocyte dysfunction resulted from ERK hyperactivation and dysregulated autophagy, observed as accumulation of vesicles, vacuolar structures, and degenerated mitochondria. The diminished autophagic flux in SPRED2-/- hearts was reflected by a reduced LC3-II/LC3-I ratio and by decreased Atg7, Atg4B and Atg16L expression. Furthermore, the autophagosomal adaptors p62/SQSTM1 and NBR1 and lysosomal Cathepsin D accumulated in SPRED2-/- hearts. In wild-type hearts, SPRED2 interacted physically with p62/SQSTM1, NBR1, and Cathepsin D, indicating that SPRED2 is required for autophagolysosome formation in regular autophagy. Restored inhibition of MAPK signaling by selumetinib led to an increase in autophagic flux in vivo. Therefore, our study identifies SPRED2 as a novel, indispensable regulator of cardiac autophagy. Vice versa, SPRED2 deficiency impairs autophagy, leading to cardiac dysfunction and life-threatening arrhythmias.

Impact of tissue photon attenuation in small animal cardiac PET imaging.

OBJECTIVES: Tissue photon attenuation is one of the essential artifacts requiring correction in clinical cardiac positron emission tomography (PET) imaging. However, due to small body size its impact on diagnostic accuracy in small rodents is considered to be limited or even ignorable. The present cardiac PET study compares lean and obese rats to determine the influence of tissue attenuation on quantitative assessment as well as regional tracer distribution. METHODS: A dedicated small animal PET system equipped with a 57Co rotating source for transmission was used. To assess the impact of tissue attenuation in rats with different body sizes, cardiac 18F-FDG -PET studies for Zucker diabetic fatty rats (obese rats) and Zucker lean rats (lean rats) were performed. The radiotracer activity reduction by attenuation was compared between the two groups. Regional tracer distribution calculated with and without attenuation correction was also assessed. RESULTS: The chest diameter was significantly longer in obese than in lean rats (5.6±0.3cm in obese and 4.5±0.2cm in lean rats, p<0.0001). Whereas the activity reduction by attenuation was significantly greater in obese than in lean rats (44.1±2.5% and 5.1±3.1%, p<0.0001), the regional variation of tissue attenuation among the ventricular walls was minimal in both lean (p=0.73) and obese rats (p=0.65). CONCLUSION: Attenuation correction is indispensable for accurate comparison of cardiac tracer activity between animals with different body size, whereas it can be omitted for evaluation of regional tracer distribution.

CD4+ Foxp3+ T-cells contribute to myocardial ischemia-reperfusion injury.

OBJECTIVE: The present study analyzed the effect of CD4+ Forkhead box protein 3 negative (Foxp3-) T-cells and Foxp3+ CD4+ T-cells on infarct size in a mouse myocardial ischemia-reperfusion model. APPROACH AND RESULTS: We examined the infarct size as a fraction of the area-at-risk as primary study endpoint in mice after 30minutes of coronary ligation followed by 24hours of reperfusion. CD4+ T-cell deficient MHC-II KO mice showed smaller histologically determined infarct size (34.5±4.7% in MHCII KO versus 59.4±4.9% in wildtype (WT)) and better preserved ejection fraction determined by magnetic resonance tomography (56.9±2.8% in MHC II KO versus 39.0±4.2% in WT). MHC-II KO mice also displayed better microvascular perfusion than WT mice after 24hours of reperfusion. Also CD4+ T-cell sufficient OT-II mice, which express an in this context irrelevant T-cell receptor, revealed smaller infarct sizes compared to WT mice. However, MHC-II blocking anti-I-A/I-E antibody treatment was not able to reduce infarct size indicating that autoantigen recognition is not required for the activation of CD4+ T-cells during reperfusion. Flow-cytometric analysis also did not detect CD4+ T-cell activation in heart draining lymph nodes in response to 24hours of ischemia-reperfusion. Adoptive transfer of CD4+ T-cells in CD4 KO mice increased the infarct size only when including the Foxp3+ CD25+ subset. Depletion of CD4+ Foxp3+ T-cells in DEREG mice enabling specific conditional ablation of this subset by treatment with diphtheria toxin attenuated infarct size as compared to diphtheria toxin treated WT mice. CONCLUSIONS: CD4+ Foxp3+ T-cells enhance myocardial ischemia-reperfusion injury. CD4+ T-cells exert injurious effects without the need for prior activation by MHC-II restricted autoantigen recognition.

Inhibition of Platelet GPVI Protects Against Myocardial Ischemia-Reperfusion Injury.

OBJECTIVE: The objective of this study was to investigate the effects of platelet inhibition on myocardial ischemia-reperfusion (IR) injury. APPROACH AND RESULTS: Timely restoration of coronary blood flow after myocardial infarction is indispensable but leads to additional damage to the heart (myocardial IR injury). Microvascular dysfunction contributes to myocardial IR injury. We hypothesized that platelet activation during IR determines microvascular perfusion and thereby the infarct size in the reperfused myocardium. The 3 phases of thrombus formation were analyzed by targeting individual key platelet-surface molecules with monoclonal antibody derivatives: (1) adhesion (anti-glycoprotein [GP]-Ib), (2) activation (anti-GPVI), and (3) aggregation (anti-GPIIbIIIa) in a murine in vivo model of left coronary artery ligation (30 minutes of ischemia followed by 24 hours of reperfusion). Infarct sizes were determined by Evans Blue/2,3,5-triphenyltetrazolium chloride staining, infiltrating neutrophils by immunohistology. Anti-GPVI treatment significantly reduced infarct size versus control, whereas anti-GPIb or anti-GPIIbIIIa antibody fragments showed no significant differences. Mechanistically, anti-GPVI antibody-mediated reduction of infarct size was not because of impaired Ca(2+) signaling or platelet degranulation because mice deficient in store-operated calcium channels (stromal interaction molecule 1, ORAI1), α-granules (Nbeal2(-/-)), and dense granule release (Unc13d(-/-)) had similar infarct sizes as control animals. Protective effects of anti-GPVI treatment were accompanied by improved microperfusion. Leukocyte infiltration was reduced in both anti-GPVI and anti-GPIb-treated IR mice. CONCLUSIONS: Inhibition of platelet activation by an anti-GPVI antibody, but not inhibition of platelet adhesion or aggregation by an anti-GPIb or anti-GPIIbIIIa antibody significantly reduces infarct size. The reduction of the infarct size is primarily based on an improved microperfusion after anti-GPVI antibody treatment.