The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN) response signatures in tubular cells and in keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous, and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histological differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.

Abstract-In ventricular cardiac myocytes, T-tubule density is an important determinant of the synchrony of sarcoplasmic reticulum (SR) Ca 2ϩ release and could be involved in the reduced SR Ca 2ϩ release in ischemic cardiomyopathy. We therefore investigated T-tubule density and properties of SR Ca 2ϩ release in pigs, 6 weeks after inducing severe stenosis of the circumflex coronary artery (91Ϯ3%, Nϭ13) with myocardial infarction (8.8Ϯ2.0% of total left ventricular mass). Severe dysfunction in the infarct and adjacent myocardium was documented by magnetic resonance and Doppler myocardial velocity imaging. Myocytes isolated from the adjacent myocardium were compared with myocytes from the same region in weight-matched control pigs. T-tubule density quantified from the di-8-ANEPPS (di-8-butyl-aminonaphthyl-ethylene-pyridinium-propyl-sulfonate) sarcolemmal staining was decreased by 27Ϯ7% (PϽ0.05). Synchrony of SR Ca 2ϩ release (confocal line scan images during whole-cell voltage clamp) was reduced in myocardium myocytes. Delayed release (ie, ] i occurring later than 20 ms) occurred at 35.5Ϯ6.4% of the scan line in myocardial infarction versus 22.7Ϯ2.5% in control pigs (PϽ0.05), prolonging the time to peak of the line-averaged [Ca 2ϩ ] i transient (121Ϯ9 versus 102Ϯ5 ms in control pigs, PϽ0.05). Delayed release colocalized with regions of T-tubule rarefaction and could not be suppressed by activation of protein kinase A. The whole-cell averaged [Ca 2ϩ ] i transient amplitude was reduced, whereas L-type Ca 2ϩ current density was unchanged and SR content was increased, indicating a reduction in the gain of Ca 2ϩ -induced Ca 2ϩ release. In conclusion, reduced T-tubule density during ischemic remodeling is associated with reduced synchrony of Ca 2ϩ release and reduced efficiency of coupling Ca 2ϩ influx to Ca Key Words: myocardial infarction Ⅲ contractility Ⅲ myocytes Ⅲ calcium A lthough new therapeutic approaches have decreased the mortality associated with myocardial infarction (MI) over the past decades, 1 many patients nevertheless sustain a regional loss of myocardial contractile tissue following an ischemic event. The resulting increased hemodynamic burden on the left ventricle leads to structural and functional changes in the remaining viable myocardium, which further reduces ventricular performance, a process referred to as myocardial remodeling. 2 Sustained regional chronic and/or intermittent ischemia further contributes to this process, and the resulting ischemic cardiomyopathy is currently among the major causes of heart failure. 3 Contractile dysfunction of the ventricle is partly related to the abnormal loading in vivo 4 and partly to the intrinsic properties of the cardiomyocytes. Myocytes isolated from patients with ischemic cardiomyopathy at the time of heart transplantation have a reduced contractile function resulting from abnormal Ca 2ϩ handling. [5][6][7] Animal models have examined the mechanisms of cellular dysfunction in ischemic cardiomyopathy in more detail. Myocytes from the infarct border...

The secondary structure of the P5abc subdomain (a 56-nt RNA) of the Tetrahymena thermophila group I intron ribozyme has been determined by NMR. Its base pairing in aqueous solution in the absence of magnesium ions is significantly different from the RNA in a crystal but is consistent with thermodynamic predictions. On addition of magnesium ions, the RNA folds into a tertiary structure with greatly changed base pairing consistent with the crystal structure: three Watson-Crick base pairs, three G⅐U base pairs, and an extra-stable tetraloop are lost. The common assumption that RNA folds by first forming secondary structure and then forming tertiary interactions from the unpaired bases is not always correct.RNA plays many roles in biological processes, and our knowledge of its importance is still expanding rapidly (1, 2). By understanding how RNA folds into its three-dimensional structure, we can begin to predict its folding from its sequence and can greatly improve our understanding of its biological functions. The standard RNA folding mechanism is a two-step process. The RNA first folds into a secondary structure, which then folds into a three-dimensional tertiary structure stabilized by interactions between the preformed secondary structural motifs (3-7). Divalent metal ions bound at a few specific locations appear to be crucial for the tertiary folding (8, 9). RNA secondary structures can be deduced from a phylogenetic comparison of many related sequences or from a calculation of free energies (based on thermodynamic measurements of model oligonucleotides) for stable secondary structures. Secondary structures and low-resolution tertiary structures can also be obtained from biochemical experiments that reveal nucleotide accessibility and detect long-range interactions. However, NMR spectroscopy and x-ray diffraction are the major tools for high-resolution structure determination of RNA.The crystal structure of the P4-P6 domain of the Tetrahymena group I intron at high resolution (10-12) has revealed a tremendous amount of new information on RNA structure and folding. When the Tetrahymena group I intron (414 nt) folds into a catalytically active form, the 160-nt P4-P6 domain is the first to fold (6,13,14). Cleavage experiments with Fe-EDTA showed that the isolated domain and the domain in the intron fold into the same tertiary structure on addition of millimolar concentrations of magnesium ions (15). A cluster of five Mg 2ϩ were found coordinated to phosphate oxygen atoms in the A-rich bulge and in the 3-helix junction of the P5abc subdomain of the P4-P6 domain (10, 11). To confirm the identities of the metal-coordinated phosphate groups, phosphorothioate substitution experiments were performed on the 160-nt P4-P6 domain and a 56-nt P5abc subdomain (9). Native gel electrophoresis experiments separated two forms of the RNA molecules: folded and unfolded. By systematically replacing sulfur atoms for oxygen atoms, seven pro-Rp phosphate oxygens were found to be necessary for tertiary folding in solution in bo...

The stabilities and structures of a series of RNA octamers containing symmetric tandem mismatches were studied by UV melting and imino proton NMR. The free energy increments for tandem mismatch formation are found to depend upon both mismatch sequence and adjacent base pairs. The observed sequence dependence of tandem mismatch stability is UGGU > GUUG > GAAG > or = AGGA > UUUU > CAAC > or = CUUC approximately UCCU approximately CCCC approximately ACCA approximately AAAA, and the closing base pair dependence is 5'G3'C > 5'C3'G > 5'U3'A approximately 5'A3'U. These results differ from expectations based on models used in RNA folding algorithms and from the sequence dependence observed for folding of RNA hairpins. Imino proton NMR results indicate the sequence dependence is partially due to hydrogen bonding within mismatches.

The three-dimensional solution structure of the RNA self-complementary duplex [sequence: see text] was derived from two-dimensional NMR and the iterative relaxation matrix approach. Each GA mismatch forms two hydrogen bonds: A-NH6 to G-O6 and A-N1 to G-NH1 (imino). This is the first three-dimensional RNA structure with imino hydrogen-bonded tandem GA mismatches. This GA structure is totally different from the sheared tandem GA structure in [sequence: see text] which also has two hydrogen bonds: A-N7 to G-NH2 and A-NH6 to G-N3 [SantaLucia, J., Jr., & Turner, D. H. (1993) Biochemistry 32, 12612-12623]. In particular, the sheared and imino GA mismatches produce a narrowing and widening of the backbone, respectively. The results show that substitutions of Watson-Crick base pairs can have dramatic effects on the three-dimensional structures of adjacent non-Watson-Crick paired regions; i.e., the structure depends on sequence context. Thus compensating substitutions in site-directed mutagenesis experiments may not always restore biological activities.

UV melting and imino proton NMR studies show that the stabilities and structures of tandem GA mismatches in RNA are dependent upon the closing base pairs around these mismatches. Internal loops of sequence 5'XGAY3'3'YAGXS' and 5'XAGY3'3'YGAX5' in the middle of octanucleotides have a range of stabilities over 5 kcal/mol when XY is a Watson-Crick or GU pair. The order of stabilities for these internal loops is 5'-GGAC-3' > UGAG, CGAG > AGAU > UGAA > GGAU. The motifs GGAC, UGAG, and CGAG are stabilizing, while the other GA motifs are destabilizing. The GAGC motif is more stable than CAGG and CGAG, but less stable than GGAC. Chemical shifts for imino protons suggest that the G imino proton of each GA mismatch in 5'-GGAC-3', 5'-GAGC-3', and 5'-CAGG-3' [SantaLucia, J., Jr., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819] is involved in a hydrogen bond to the base A, whereas in other 5'-XGAY-3' sequences, it is not involved in a hydrogen bond to the base A.

Background: Cardiac fibroblasts (CF) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Due to the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is still missing. The purpose of this study was to define the CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate their function. Methods: Collagen1α1-GFP + CF were characterized after myocardial infarction (MI) by single-cell and bulk RNA-seq, ATAC-seq and functional assays. Swine and patient samples were studied using bulk RNA-seq. Results: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear pro-fibrotic signature, express high levels of Collagen Triple Helix Repeat Containing 1 ( Cthrc1 ) and localize into the scar. Non-canonical TGF-β signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Moreover, the absence of CTHRC1 results in pronounced lethality due to ventricular rupture. Finally, a population of CF with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. Conclusions: We report CF heterogeneity, their dynamics during the course of MI and redefine the CF that respond to cardiac injury and participate in myocardial remodeling. Our study identifies Cthrc1 as a novel regulator of the healing scar process, and as a target for future translational studies.

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