Lipofuscin is an intracellular aggregate of highly oxidized proteins that cannot be digested in the ubiquitin-proteasome system and accumulate mainly in lysosomes, especially in aged cells and pathological conditions. However, no systematic study has evaluated the cardiac accumulation of lipofuscin during human ageing and sudden cardiac death (SCD). Age estimation in unidentified bodies and postmortem SCD diagnosis are important themes in forensics. Thus, we aimed to elucidate their correlations with myocardial lipofuscin accumulation. We collected 76 cardiac samples from autopsy patients aged 20–97 years. After histopathological examination, myocardial lipofuscin was measured using its autofluorescence. Lipofuscin accumulated mainly in the perinuclear zone, and its accumulation rate positively correlated with chronological ageing (r = 0.82). Meanwhile, no significant change in lipofuscin level was observed with different causes of death, including SCD. There was also no significant change in lipofuscin level in relation to body mass index, serum brain natriuretic peptide level, or heart weight. Moreover, we performed LC3 and p62 immunoblotting to evaluate autophagic activity, and no change was observed in ageing. Therefore, lipofuscin accumulation more directly reflects chronological ageing rather than human cardiac pathology. Our study reveals the stability and utility of cardiac lipofuscin measurement for age estimation during autopsy.

Although myo-inositol pyrophosphates such as diphosphoinositol pentakisphosphate (InsP) are important in biology, little quantitative information is available regarding their presence in mammalian organisms owing to the technical difficulties associated with accurately detecting these materials in biological samples. We have developed an analytical method whereby InsP and its precursor inositol hexakisphosphate (InsP) are determined directly and sensitively using tandem mass spectrometry coupled with hydrophilic interaction liquid chromatography (HILIC). InsP and InsP peak symmetry is influenced greatly by the buffer salt composition and pH of the mobile phase used in HILIC analysis. The use of 300 mM ammonium carbonate (pH 10.5) as an aqueous mobile phase resolves InsP and InsP on a polymer-based amino HILIC column with minimal peak tailing. Method validation shows that InsP and InsP can be quantitated from 20-500 pmol with minimal intra-day/inter-day variance in peak area and retention time. The concentration of InsP in C57BL/6J mouse brain (40.68 ± 3.84 pmol/mg wet weight) is successfully determined. HILIC‒MS/MS analysis using HEK293 culture cells confirms previous observations that InsP is induced by NaF treatment and ectopic expression of InsPK2, a primary kinase for InsP synthesis. Furthermore, this analysis reveals the abundance of InsP (50.46 ± 18.57 pmol/10 cells) and scarcity of InsP in human blood cells. The results demonstrate that HILIC‒MS/MS analysis can quantitate endogenous InsP and InsP in mouse and human samples, and we expect that the method will contribute to further understanding of InsP functions in mammalian pathobiology.

The study aims to clarify the mechanism in patients with neurally mediated syncope (NMS), focusing on the adenylate cyclase (AC) activity level in lymphocytes. This study included 40 subjects: 22 healthy volunteers and 18 NMS patients. We investigated the changes in AC activity that occur during of syncope at rest and during the head-up tilt (HUT) test. We obtained 8 mL of blood at rest time and four times during the HUT test. Then, we measured the AC activity and the test reagent was added to the lymphocytes (10,000) and reacted for 30 min at room temperature. We were able to determine the standard value of AC activity when adrenaline (AD) and isoproterenol (IP) were added to lymphocytes. The results of our study showed one of the causes of NMS has a difference in AC activity level and classification of the patients into two different types of NMS was possible: either the vasodepressor type (VT) or mixed type (MT). At rest time, VT patients showed significantly higher AC activity (AD; 100 μM: p = 0.005, IP; 50 μM: p = 0.02) and MT patients showed significantly lower AC activity (AD; 10 μM: p = 0.02, IP; 50 μM: p = 0.004) than the average AC activity in healthy volunteers. Moreover, VT patients had significantly higher AC activity than healthy volunteers at the four points of the HUT test. MT patients had significantly lower AC activity (AD: p = 0.04 and IP: p = 0.04) than healthy volunteers at the rest time of HUT. Our study showed a significant difference in AC activities between NMS patients and healthy volunteers at rest. Therefore, a detailed NMS diagnosis can be made by examining AC activity levels in blood taken at rest time.

A disintegrin and metalloprotease 17 (ADAM17) is the major sheddase that processes more than 80 substrates, including tumour necrosis factor-α (TNFα). The homozygous genetic deficiency of ADAM17 causing a complete loss of ADAM17 expression was reported to be linked to neonatal inflammatory skin and bowel disease 1 (NISBD1). Here we report for the first time, a family with NISBD1 caused by functionally confirmed compound heterozygous missense variants of ADAM17, namely c.1699T>C (p.Cys567Arg) and c.1799G>A (p.Cys600Tyr). Both variants were detected in two siblings with clinical features of NISBD1, such as erythroderma with exudate in whole body, recurrent skin infection and sepsis and prolonged diarrhoea. In a cell-based assay using Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17−/− mEFs) exogenously expressing each of these mutants, phorbol 12-myristate 13-acetate-stimulated shedding was strongly reduced compared with wild-type ADAM17. Thus, in vitro functional assays demonstrated that both missense variants cause the loss-of-function of ADAM17, resulting in the development of NISBD1. Our study further expands the spectrum of genetic pathology underlying ADAM17 in NISBD1 and establishes functional assay systems for its missense variants.

Cathepsins are the major lysosomal proteases that maintain intracellular homeostasis. Herein, we investigated the alterations in myocardial cathepsin expression during aging, cardiac hypertrophy, and sudden cardiac death (SCD). Cardiac tissue and blood were sampled from autopsy cases. Subjects were classified into three groups: SCD with cardiac hypertrophy (SCH), compensated cardiac hypertrophy (CCH), and control. Immunoblotting was performed for the major cardiac cathepsins and their targets: cathepsin B, D, and L (CTSB/D/L), p62, ATP synthase subunit c (ATPSC), and α-synuclein (ASNC). Immunohistochemical analysis and ELISA using serum samples were performed for CTSD. Cardiac CTSB and CTSD were upregulated with age (r = 0.63 and 0.60, respectively), whereas the levels of CTSL, p62, ATPSC, and ASNC remained unchanged. In age-matched groups, cardiac CTSD was significantly downregulated in SCH (p = 0.006) and CTSL was moderately downregulated in CCH (p = 0.021); however, p62, ATPSC, and ASNC were not upregulated in cardiac hypertrophy. Immunohistochemistry also revealed decreased myocardial CTSD levels in SCH, and serum CTSD levels were relatively lower in SCH cases. Overall, these results suggest that upregulation of cardiac CTSB and CTSD with age may compensate for the elevated proteolytic demand, and that downregulation of CTSD is potentially linked to SCH. OPEN ACCESS Citation: Kakimoto Y, Sasaki A, Niioka M, Kawabe N, Osawa M (2020) Myocardial cathepsin D is downregulated in sudden cardiac death. PLoS ONE 15(3): e0230375. https://doi.org/10.