Readiness of Advance Care Planning Among Patients With Cardiovascular Disease.

Background: Advance care planning (ACP) is a widely advocated strategy to improve outcomes at end-of-life care for patients suffering from heart failure (HF). However, finding the right time to start ACP is challenging for healthcare providers because it is often a sensitive issue for patients with HF and their families. We interviewed patients with cardiovascular diseases regarding ACP readiness and investigated the relationship between the ACP desire and multiple clinical prognostic parameters. Method: Eighty-one patients (average age 81.8 ± 10.3 years old, 42 men, 62 cases of HF) who introduced cardiac rehabilitation were inquired about previous ACP experience, a desire for ACP, understanding of their cardiovascular diseases, and lifestyle-associated questionnaires. Multiple logistic regression analyses were employed to identify the clinical parameters associated with ACP desire. Patients who desired ACP were also asked about their preferences for medical care at the end-of-life. Results: Nine patients (11.1%) had previous experience with ACP, and 28 (34.6%) preferred to implement ACP. Patients who did not want to implement ACP were 54.3%. Patients with HF showed a higher acceptance rate of ACP (odds ratio [OR] 5.56, p = 0.015). Interestingly, patients harboring skeletal muscle frailty showed lower ACP acceptance, while patients with non-frailty rather positively wanted to implement ACP. Two types of prognosis evaluation scales, such as the Enhanced Feedback for Effective Cardiac Treatment (EFFECT) risk score and the Japanese Version of Supportive and Palliative Care Indicators Tool (SPICT-JP), identified 31 patients (38.3%) needing ACP; however, 19 (61.3%) did not want ACP. The wish not to attempt resuscitation and life-prolonging treatment at the end-of-life reached approximately 70% among patients who requested ACP. Conclusions: Although patients with HF tended to be ready for implementing ACP, the presence of skeletal muscle frailty was negatively associated with ACP preference. Indeed, patients who should be considered ACP were not carried out and did not desire it. Earlier introduction of ACP into patients before having skeletal muscle frailty may be considered.

Genetic and molecular analysis of wild-derived arrhythmic mice.

A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus). This variant was characterized by an initial maintenance of damped oscillations and subsequent loss of rhythmicity after being transferred from light-dark (LD) cycles to constant darkness (DD). To map the genes responsible for the persistence of rhythmicity (circadian ratio) and the length of free-running period (tau), quantitative trait locus (QTL) analysis was performed using F(2) mice obtained from an F(1) cross between the circadian variant and C57BL/6J mice. As a result, a significant QTL with a main effect for circadian ratio (Arrhythmicity; Arrh-1) was mapped on Chromosome (Chr) 8. For tau, four significant QTLs, Short free-running period (Sfp-1) (Chr 1), Sfp-2 (Chr 6), Sfp-3 (Chr 8), Sfp-4 (Chr 11) were determined. An epistatic interaction was detected between Chr 3 (Arrh-2) and Chr 5 (Arrh-3). An in situ hybridization study of clock genes and mouse Period1::luciferase (mPer1::luc) real-time monitoring analysis in the suprachiasmatic nucleus (SCN) suggested that arrhythmicity in this variant might not be attributed to core circadian mechanisms in the SCN neurons. Our strategy using wild-derived variant mice may provide a novel opportunity to evaluate circadian and its related disorders in human that arise from the interaction between multiple variant genes.

Reorganization of the suprachiasmatic nucleus coding for day length.

In mammals, the suprachiasmatic nucleus (SCN), the circadian pacemaker, receives light information via the retina and functions in the entrainment of circadian rhythms and in phasing the seasonal responses of behavioral and physiological functions. To better understand photoperiod-related alterations in the SCN physiology, we analyzed the clock gene expression in the mouse SCN by performing in situ hybridization and real-time monitoring of the mPer1::luc bioluminescence. Under long photoperiod (LP) conditions, the expression rhythms of mPer1 and Bmal1 in the caudal SCN phase-led those in the rostral SCN; further, within the middle SCN, the rhythms in the ventrolateral (VL)-like subdivision advanced compared with those in the dorsomedial (DM)-like subdivision. The mPer1::luc rhythms in the entire coronal slice obtained from the middle SCN exhibited 2 peaks with a wide peak width under LP conditions. Imaging analysis of the mPer1::luc rhythms in several subdivisions of the rostral, middle, caudal, and horizontal SCN revealed wide regional variations in the peak time in the rostral half of the SCN under LP conditions. These variations were not due to alterations in the waveform of a single SCN neuronal rhythm. Our results indicate that LP conditions induce phase changes in the rhythms in multiple regions in the rostral half of the SCN; this leads to different circadian waveforms in the entire SCN, coding for day length.

Bimodal clock gene expression in mouse suprachiasmatic nucleus and peripheral tissues under a 7-hour light and 5-hour dark schedule.

Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of mPer1 bioluminescence on each side of the SCN, in parallel with maintaining synchronization between the left and right sides of the SCN under an artificial light:dark:light:dark (LDLD) 7:5:7:5 condition. In situ hybridization analysis of mPer1 and mBmal1 mRNA distribution in the SCN showed that in 1 photophase (morning photophase; M) of LDLD, the mPer1 level in the ventrolateral-like (VL-like) subdivision of the SCN was higher than that in the dorsomedial-like (DM-like) subdivision, and this regional distribution pattern was reversed in another photophase (evening photophase; E). In contrast, the mBmal1 level was higher in the DM-like subdivision than in the VL-like subdivision in the M phase, and this distribution changed in the E phase. The prokineticin 2 (PK2) mRNA that encodes an SCN output molecule that is thought to transmit the circadian locomotor rhythms was reduced in both the DM-like and VL-like SCN and did not clearly correlate with the activity under the LDLD condition. The expression of mPer1 and mPer2 in the liver was clearly bimodal, whereas the expressions of other clock genes were not synchronized to the LDLD condition. These results may provide important insights into the mechanism underlying the splitting or bimodal rhythms that may in turn facilitate the understanding of the ability to measure the seasonal day length in mammals.

Parentally imprinted allele typing at a short tandem repeat locus in intron 1a of imprinted gene KCNQ1.

A short tandem repeat (STR) in the intron 1a of paternally imprinted gene, KCNQ1, is evaluated as a new probe for use in parentally imprinting allele (PIA) typing. This typing can determine the inheritance of one allele from father by the methylation difference. Allelic and genotypic frequencies of the STR were determined using samples from 175 unrelated Japanese and 170 unrelated Germans. The polymorphism information contents were 0.652 and 0.634 for the Japanese and the Germans, respectively, indicating usefulness in individual identification. This method was applied to five Japanese families consisting of 19 individuals. Genomic DNA was digested by methylation-sensitive restriction endonucleases, HhaI and HapII, followed by PCR amplification using two-step sandwich primer sets and the products were analyzed on polyacrylamide gel electrophoresis. For all of the families, each child's paternal allele given by PIA typing corresponded to one of the two alleles from father, not the two from mother, that were determined by the STR genotyping. The results demonstrate that this STR probe is feasible for use in PIA typing and that its typing method can contribute to paternity testing.

Applicability of the parentally imprinted allele (PIA) typing of a VNTR upstream the H19 gene to forensic samples of different tissues.

The parentally imprinted allele (PIA) typing that we have recently developed determines parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene. The usefulness of this typing was demonstrated by its application to blood samples in paternity cases. However, its applicability to other tissue DNA remains to be tested. DNA samples from fifteen different postmortem tissues such as cerebrum, skeletal muscle and skin were examined, all of which were obtained from three autopsy cases 2-11h after death. DNA was digested with a methylation-sensitive HhaI enzyme and diluted solutions of the digests were subjected to the first PCR amplification, providing amplification of only the paternal H19 methylated allele. Subsequent VNTR typing was carried out for the amplified products to determine which allele was of paternal origin. No tissue-dependent difference was observed and all the samples examined, though degraded, were successfully used for determining the paternal allele. These results substantiate the usefulness of PIA typing in forensic examinations. Its application to two identity cases, a burned male body and a male body with adipocere formation, was also shown.

Novel paternity testing by distinguishing parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene.

Conventional PCR-based genotyping is useful for forensic testing but cannot be used to determine parental origins of alleles in DNA specimens. Here we describe a novel method of combined conventional genotyping and PIA typing (parentally imprinted allele typing) at a minisatellite region upstream from the H19 locus. The PIA typing uses two sets of primers and DNA digested with methylation-sensitive Hha I enzyme. The first amplification produces only the methylated fragment of paternal H19 allele, and the second detects polymorphism in the minisatellite. Hence, this distinguishes paternal and maternal alleles by difference in the DNA methylation. Furthermore, the polymorphism in this polymorphic locus was examined using 199 unrelated Japanese and 171 unrelated Germans, their polymorphism information content being 0.671 and 0.705, respectively. Feasibility of this typing is demonstrated for six families, and the usefulness is shown by application to paternity testing.