Aims Inadequate healing after myocardial infarction (MI) leads to heart failure and fatal ventricular rupture, while optimal healing requires timely induction and resolution of inflammation. This study tested the hypothesis that heat shock protein B1 (HSPB1), which limits myocardial inflammation during endotoxemia, modulates wound healing after MI. Methods and results To test this hypothesis, cardiomyocyte-specific HSPB1 knockout (Hspb1-/-) mice were generated using the Cre-LoxP recombination system. MI was induced by ligation of the left anterior descending coronary artery in Hspb1-/- and wild-type (WT) littermates. HSPB1 was up-regulated in cardiomyocytes of WT animals in response to MI, and deficiency of cardiomyocyte HSPB1 increased MI-induced cardiac rupture and mortality within 21 days after MI. Serial echocardiography showed more aggravated remodelling and cardiac dysfunction in Hspb1-/- mice than in WT mice at 1, 3, and 7 days after MI. Decreased collagen deposition and angiogenesis, as well as increased MMP2 and MMP9 activity, were also observed in Hspb1-/- mice compared with WT controls after MI, using immunofluorescence, polarized light microscopy, and zymographic analyses. Notably, Hspb1-/- hearts exhibited enhanced and prolonged leucocyte infiltration, enhanced expression of inflammatory cytokines, and enhanced TLR4/MyD88/NFκB activation compared with WT controls after MI. In-depth molecular analyses in both mice and primary cardiomyocytes demonstrated that cardiomyocyte-specific knockout of HSPB1 increased nuclear factor-κB (NFκB) activation, which promoted the expression of proinflammatory mediators. This led to increased leucocyte recruitment, thereby to excessive inflammation, ultimately resulting in adverse remodelling, cardiac dysfunction, and cardiac rupture following MI. Conclusion These data suggest that HSPB1 acts as a negative regulator of NFκB-mediated leucocyte recruitment and the subsequent inflammation in cardiomyocytes. Cardiomyocyte HSPB1 is required for wound healing after MI and could be a target for myocardial repair in MI patients.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18095号 / 理博第3973号 / 新制||理||1573(附属図書館) / 30953 / 京都大学大学院理学研究科化学専攻 / (主査)教授 三木 邦夫, 教授 杉山 弘, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18514号 / 医博第3934号 / 新制||医||1006(附属図書館) / 31400 / 京都大学大学院医学研究科医学専攻 / (主査)教授 上本 伸二, 教授 三森 経世, 教授 長田 重一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21339号 / 医博第4397号 / 新制||医||1031(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 宮本 享, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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A Chlamydia trachomatis heat shock protein 60kDa (chsp60) exhibits increased expression in response to iron limitation. Genome sequencing revealed three genes encoding chsp60s. The objective of this study was to generate peptide antisera that would selectively recognize each chsp60. The DNA sequence for each C. trachomatis serovar E chsp60 was determined and compared with existing genome sequences. Predictive amino acid sequences were evaluated for peptides unique to each chsp60. Synthetic peptides were used to generate antisera; the resultant sera were purified by affinity chromatography and adsorbed to reduce cross-reactivity and increase monospecificity. Antisera were evaluated against each recombinant chsp60 protein by Western blotting. Reactivity against native chsp60s was visualized by transmission electron microscopy. Initial experiments indicate that expression of the second chsp60 (encoded by groEL_2) is increased during iron limitation. The production of chsp60 antibodies in human patients is associated with damaging sequelae in chlamydial genital and ocular infections.

Chlamydomonas priscuii is a psychrophilic green alga found 17 m below the permanently ice-covered surface of the Antarctic Lake Bonney, where it experiences a myriad of extreme environmental conditions, including low temperature, low light, and high salinity. While this habitat is extreme, it is also very stable, and this alga rarely experiences changes in its environment. Heat shock proteins (HSPs) are a ubiquitous family of chaperone proteins that perform important housekeeping and stress-related roles. In most organisms, including the model green alga Chlamydomonas reinhardtii, HSP expression is induced during abiotic stress to regain protein homeostasis – a process regulated by heat shock transcription factors (HSFs). This work shows that C. priscuii constitutively accumulates high protein levels of HSPs in steady-state conditions but fails to induce additional HSP accumulation during heat and low temperature, high and low salt, high light, and with canavanine treatment. In this study, a single HSF was identified in the C. priscuii genome. Comparative sequence analysis revealed that most domains characteristic of a functional HSF are conserved, but the expression of a full length HSF1 transcript could not be detected in the cell. Furthermore, the promoters of many C. priscuii HSPs lack binding sites for HSF. This work has shown that C. priscuii has a diminished ability to regulate HSP expression under stressful conditions, which we hypothesize is a result of life in an extreme but very stable environment. This is the first demonstration of a loss of HSP accumulation in green algae, which carries implications on the ability of psychrophiles to survive in the face of climate change.

Cardiovascular disease (CVD) is the leading cause of death globally, and tissue ischemia induces disorders including myocardial infarction, pulmonary arterial hypertension, and atherosclerosis. Ischemic conditions considerably alter cellular homeostasis as well as metabolism and can result in cardiovascular dysfunction. Therefore, there is a dire need to develop a novel treatment strategy for curing myocardial ischemia via manipulating cellular networking and metabolism. Furthermore, studies have shown that ischemic conditions induce cellular stress such that it modifies the expression of various cellular proteins and may even promote cell death. Cellular proteins must fold into a three-dimensional, native state to become functional. To ensure efficient folding and prevent aggregation, molecular chaperones assist with the folding/refolding process. Additionally, chaperones regulate cellular proteostasis through the ubiquitin-proteosome system (UPS) or autophagy. The heat-shock protein (HSP) is a molecular chaperone family upregulated during stress conditions to assist cells with proper protein folding, stability, and turnover. Specifically, the 70-kDa heat-shock protein (Hsp70) has numerous cytoprotective and immunomodulatory effects through its interaction with components of several cellular pathways. This interaction is dependent on nucleotide exchange factors (NEFs) to hold the open conformation of HSP70's nucleotide-binding domain (NBD). Proteins in the Bcl-2- associated athanogene (BAG) family are a group of co-chaperones that interact with the ATPase domain of HSP70 and help maintain homeostasis. The BAG family proteins (BAG1-6) share an evolutionarily conserved region at their C-termini (the BAG domain), and BAG5 specifically is unique in that it contains five of these domains. BAG5 plays a crucial role in maintaining cellular homeostasis and viability. This project explores the role of BAG5 as a co-chaperone and potential therapeutic tool to improve cardiomyocyte function under ischemic conditions.