Single-Cell Transcriptomic Analysis Reveals Myocardial Fibrosis Mechanism of Doxorubicin-Induced Cardiotoxicity.

Myocardial fibrosis is a pathological feature of doxorubicin-induced chronic cardiotoxicity that severely affects the prognosis of oncology patients. However, the specific cellular and molecular mediators driving doxorubicin-induced cardiac fibrosis, and the relative impact of different cell populations on cardiac fibrosis, remain unclear.This study aimed to explore the mechanism of doxorubicin-induced cardiotoxicity and myocardial fibrosis and to find potential therapeutic targets. Single-cell RNA sequencing was used to analyze the transcriptome of non-cardiomyocytes from normal and doxorubicin-induced chronic cardiotoxicity in mouse model heart tissue.We established a mouse model of doxorubicin-induced cardiotoxicity with a well-defined fibrotic phenotype. Analysis of single-cell sequencing results showed that fibroblasts were the major origin of extracellular matrix in doxorubicin-induced myocardial fibrosis. Further resolution of fibroblast subclusters showed that resting fibroblasts were converted to matrifibrocytes and then to myofibroblasts to participate in the myocardial remodeling process in response to doxorubicin treatment. Ctsb expression was significantly upregulated in fibroblasts after doxorubicin-induced.This study provides a comprehensive map of the non-cardiomyocyte landscape at high resolution, reveals multiple cell populations contributing to pathological remodeling of the cardiac extracellular matrix, and identifies major cellular sources of myofibroblasts and dynamic gene-expression changes in fibroblast activation. Finally, we used this strategy to detect potential therapeutic targets and identified Ctsb as a specific target for fibroblasts in doxorubicin-induced myocardial fibrosis.

Single-cell transcriptomic identified HIF1A as a target for attenuating acute rejection after heart transplantation.

Acute rejection (AR) is an important contributor to graft failure, which remains a leading cause of death after heart transplantation (HTX). The regulation of immune metabolism has become a new hotspot in the development of immunosuppressive drugs. In this study, Increased glucose metabolism of cardiac macrophages was found in patients with AR. To find new therapeutic targets of immune metabolism regulation for AR, CD45+ immune cells extracted from murine isografts, allografts, and untransplanted donor hearts were explored by single-cell RNA sequencing. Total 20 immune cell subtypes were identified among 46,040 cells. The function of immune cells in AR were illustrated simultaneously. Cardiac resident macrophages were substantially replaced by monocytes and proinflammatory macrophages during AR. Monocytes/macrophages in AR allograft were more active in antigen presentation and inflammatory recruitment ability, and glycolysis. Based on transcription factor regulation analysis, we found that the increase of glycolysis in monocytes/macrophages was mainly regulated by HIF1A. Inhibition of HIF1A could alleviate inflammatory cells infiltration in AR. To find out the effect of HIF1A on AR, CD45+ immune cells extracted from allografts after HIF1A inhibitor treatment were explored by single-cell RNA sequencing. HIF1A inhibitor could reduce the antigen presenting ability and pro-inflammatory ability of macrophages, and reduce the infiltration of Cd4+ and Cd8a+ T cells in AR. The expression of Hif1α in AR monocytes/macrophages was regulated by pyruvate kinase 2. Higher expression of HIF1A in macrophages was also detected in human hearts with AR. These indicated HIF1A may serve as a potential target for attenuating AR.

Palmitoylation of the KATP channel Kir6.2 subunit promotes channel opening by regulating PIP2 sensitivity.

A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.

ß2-Adrenergic Stimulation Compartmentalizes ß1 Signaling Into Nanoscale Local Domains by Targeting the C-Terminus of ß1-Adrenoceptors.

RATIONALE: ßARs (ß-adrenergic receptors) are prototypical GPCRs (G protein-coupled receptors) that play a pivotal role in sympathetic regulation. In heart cells, ß1AR signaling mediates a global response, including both l-type Ca2+ channels in the sarcolemma/T tubules and RyRs (ryanodine receptors) in the SR (sarcoplasmic reticulum). In contrast, ß2AR mediates local signaling with little effect on the function of SR proteins. OBJECTIVE: To investigate the signaling relationship between ß1ARs and ß2ARs. METHOD AND RESULTS: Using whole-cell patch-clamp analyses combined with confocal Ca2+ imaging, we found that the activation of compartmentalized ß2AR signaling was able to convert the ß1AR signaling from global to local mode, preventing ß1ARs from phosphorylating RyRs that were only nanometers away from sarcolemma/T tubules. This offside compartmentalization was eliminated by selective inhibition of ß2AR, GRK2 (GPCR kinase-2), ßarr1 (ß-arrestin-1), and phosphodiesterase-4. A knockin rat model harboring mutations of the last 3 serine residues of the ß1AR C terminus, a component of the putative ßarr1 binding site and GRK2 phosphorylation site, eliminated the offside compartmentalization conferred by ß2AR activation. CONCLUSIONS: ß2AR stimulation compartmentalizes ß1AR signaling into nanoscale local domains in a phosphodiesterase-4-dependent manner by targeting the C terminus of ß1ARs. This finding reveals a fundamental negative feed-forward mechanism that serves to avoid the cytotoxicity of circulating catecholamine and to sharpen the transient ß1AR response of sympathetic excitation.

Sensitized signalling between L-type Ca2+ channels and ryanodine receptors in the absence or inhibition of FKBP12.6 in cardiomyocytes.

Aims: The heart contraction is controlled by the Ca2+-induced Ca2+ release (CICR) between L-type Ca2+ channels and ryanodine receptors (RyRs). The FK506-binding protein FKBP12.6 binds to RyR subunits, but its role in stabilizing RyR function has been debated for long. Recent reports of high-resolution RyR structure show that the HD2 domain that binds to the SPRY2 domain of neighbouring subunit in FKBP-bound RyR1 is detached and invisible in FKBP-null RyR2. The present study was to test the consequence of FKBP12.6 absence on the in situ activation of RyR2. Methods and results: Using whole-cell patch-clamp combined with confocal imaging, we applied a near threshold depolarization to activate a very small fraction of LCCs, which in turn activated RyR Ca2+ sparks stochastically. FKBP12.6-knockout and FK506/rapamycin treatments increased spark frequency and LCC-RyR coupling fidelity without altering LCC open probability. Neither FK506 nor rapamycin further altered LCC-RyR coupling fidelity in FKBP12.6-knockout cells. In loose-seal patch-clamp experiments, the LCC-RyR signalling kinetics, indexed by the delay for a LCC sparklet to trigger a RyR spark, was accelerated after FKBP12.6 knockout and FK506/rapamycin treatments. These results demonstrated that RyRs became more sensitive to Ca2+ triggers without FKBP12.6. Isoproterenol (1 µM) further accelerated the LCC-RyR signalling in FKBP12.6-knockout cells. The synergistic sensitization of RyRs by catecholaminergic signalling and FKBP12.6 dysfunction destabilized the CICR system, leading to chaotic Ca2+ waves and ventricular arrhythmias. Conclusion: FKBP12.6 keeps the RyRs from over-sensitization, stabilizes the potentially regenerative CICR system, and thus may suppress the life-threatening arrhythmogenesis.

Role of FK506-binding protein in Ca2+ spark regulation.

The elementary Ca2+ release events, Ca2+ sparks, has been found for a quarter of century. However, the molecular regulation of the spark generator, the ryanodine receptor (RyR) on the sarcoplasmic reticulum, remains obscure. Although each subunit of the RyR homotetramer has a site for FK506-binding protein (FKBP), the role of FKBPs in modifying RyR Ca2+ sparks has been debated for long. One of the reasons behind the controversy is that most previous studies detect spontaneous sparks, where the mixture with out-of-focus events and local wavelets prevents an accurate characterization of Ca2+ sparks. In the present study, we detected Ca2+ sparks triggered by single L-type Ca2+ channels (LCCs) under loose-seal patch clamp conditions in FK506-treated or FKBP12.6 knockout cardiomyocytes. We found that FKBP dissociation both by FK506 and by rapamycin decreased the Ca2+ spark amplitude in ventricular cardiomyocytes. This change was neither due to decreased releasable Ca2+ in the sarcoplasmic reticulum, nor explained by changed RyR sensitivity. Actually FK506 increased the LCC-RyR coupling probability and curtailed the latency for an LCC to trigger a RyR Ca2+ spark. FKBP12.6 knockout had similar effects as FK506/rapamycin treatment, indicating that the decreased spark amplitude was attributable to the dissociation of FKBP12.6 rather than FKBP12. We also explained how decreased amplitude of spontaneous sparks after FKBP dissociation sometimes appears to be increased or unchanged due to inappropriate data processing. Our results provided firm evidence that without the inter-RyR coordination by functional FKBP12.6, the RyR recruitment during a Ca2+ spark would be compromised despite the sensitization of individual RyRs.

Structural genomics studies of human caries pathogen Streptococcus mutans.

Gram-positive bacterium Streptococcus mutans is the primary causative agent of human dental caries. To better understand this pathogen at the atomic structure level and to establish potential drug and vaccine targets, we have carried out structural genomics research since 2005. To achieve the goal, we have developed various in-house automation systems including novel high-throughput crystallization equipment and methods, based on which a large-scale, high-efficiency and low-cost platform has been establish in our laboratory. From a total of 1,963 annotated open reading frames, 1,391 non-membrane targets were selected prioritized by protein sequence similarities to unknown structures, and clustered by restriction sites to allow for cost-effective high-throughput conventional cloning. Selected proteins were over-expressed in different strains of Escherichia coli. Clones expressed soluble proteins were selected, expanded, and expressed proteins were purified and subjected to crystallization trials. Finally, protein crystals were subjected to X-ray analysis and structures were determined by crystallographic methods. Using the previously established procedures, we have so far obtained more than 200 kinds of protein crystals and 100 kinds of crystal structures involved in different biological pathways. In this paper we demonstrate and review a possibility of performing structural genomics studies at moderate laboratory scale. Furthermore, the techniques and methods developed in our study can be widely applied to conventional structural biology research practice.

Ellman's reagent in promoting crystallization and structure determination of Anabaena CcbP.

Obtaining crystals presented a bottleneck in the structural study of Anabaena cyanobacterial Ca2+-binding protein (CcbP). In this report, the promoting effect of Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid); DTNB] on the crystallization of CcbP is described. CcbP contains one free cysteine. A quick and simple oxidation reaction with DTNB blocked the free cysteine in purified CcbP and generated a homogenous monomeric protein for crystallization. The crystal structure of DTNB-modified CcbP was determined by the single-wavelength anomalous diffraction method. Structure analysis indicated that DTNB modification facilitated crystallization of CcbP by inducing polar interactions in the crystal lattice. DTNB-mediated cysteine modification was demonstrated to have little effect on the overall structure and the Ca2+ binding of CcbP. Thus, DTNB modification may provide a simple and general approach for protein modification to improve the success of crystallization screening.

Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans.

Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni(2+)-chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9 Šresolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 48.97, b = 98.27, c = 101.09 Å, α = ß = γ = 90°. The optimized SMU2142 crystals diffracted to 2.7 Šresolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5 Å, α = 74.2, ß = 73.5, γ = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing.