Background-We investigated whether the improvement of cardiac function and remodeling after myocardial infarction (MI) by granulocyte colony-stimulating factor (G-CSF) relates to acceleration of the healing process, in addition to myocardial regeneration. Methods and Results-In a 30-minute coronary occlusion and reperfusion rabbit model, saline (S) or 10 g · kg Ϫ1 · d

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38␣ is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38␣ in hearts. First, we generated mice with floxed p38␣ alleles and crossbred them with mice expressing the Cre recombinase under the control of the ␣-myosin heavy-chain promoter to obtain cardiac-specific p38␣ knockout mice. These cardiac-specific p38␣ knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38␣ plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.

Myocardial infarction (MI) progresses from the acute death of myocytes and the infiltration of inflammatory cells into granulation, followed by scars. During the healing process, the myocardial interstitial cell population in the infarcted tissues increases markedly and then decreases. We postulated that apoptosis is responsible for this process. Twenty-four male Japanese white rabbits underwent a 30-minute occlusion of the left coronary artery followed by reperfusion for 2 days, 2 weeks, or 4 weeks (n=8 each). The histological features consisted of dead cardiomyocytes and marked leukocyte infiltration at 2 days after MI and granulation consisting of numerous alpha-smooth muscle actin-positive myofibroblasts, macrophage antigen-positive macrophages, and neovascularization at 2 weeks. At 4 weeks, the cellularity decreased markedly, and scars were evident. Interstitial cells with positive nick end labeling were significantly more frequent at the light microscopic level in the 2-day MI samples (5.3+/-3.6% in the center and 6.9+/-3.3% in the periphery of the infarct region) than in the 2-week (2.5+/-1.0%) and 4-week (0.5+/-0.5%) samples. DNA electrophoresis showed a clear ladder in tissues from the ischemic areas at 2 days after MI but not at 2 and 4 weeks after MI. Ultrastructurally, typical apoptotic figures, including apoptotic bodies and condensed nuclei without ruptured plasma membranes, were detected in leukocytes from all hearts with 2-day MI and in myofibroblasts, endothelial cells, and macrophages from all hearts with 2-week MI. In the electron microscopic in situ nick end labeling, immunogold particles intensely labeled the condensed chromatin of the typical apoptotic nuclei. These particles were also accumulated on nuclei of the interstitial cells showing homogeneous density but not definite condensation as typical apoptotic nuclei, suggesting an early stage of apoptosis. Thus, apoptosis plays an important role in the disappearance of both the infiltrated leukocytes and the proliferated interstitial cells after MI. This finding may have therapeutic implications for postinfarct ventricular remodeling through apoptosis handling during the healing stage of MI.

Recent consolidation of the whole-genome sequence with genome-wide transcriptome profiling revealed the existence of functional units within the genome in specific chromosomal regions, as seen in the coordinated expression of gene clusters and colocalization of functionally related genes. An efficient region-specific mutagenesis screen would greatly facilitate research in addressing the importance of these clusters. Here we use the 'local hopping' phenomenon of a DNA-type transposon, Sleeping Beauty (SB), for region-specific saturation mutagenesis. A transgenic mouse containing both transposon (acts as a mutagen) and transposase (recognizes and mobilizes the transposon) was bred for germ-cell transposition events, allowing us to generate many mutant mice. All genes within a 4-Mb region of the original donor site were mutated by SB, indicating the potential of this system for functional genomic studies within a specific chromosomal region.

In human PTCA models, a PC effect is observed in 180 s ischemia, but not in 60 s ischemia. A pharmacological PC effect is induced by NC, a KATP channel opener with a nitrate-like effect but not ISDN. This suggests that the opening of KATP channels plays an important role in the protecting effect of NC.

BackgroundCRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive.ResultsHere, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats.ConclusionsThe high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4713-y) contains supplementary material, which is available to authorized users.

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis , an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models. Electronic supplementary material The online version of this article (10.1186/s13059-019-1776-2) contains supplementary material, which is available to authorized users.

Sarpogrelate reduces the myocardial infarct size by inhibiting the serotonin release followed by enhancement of PKC-epsilon translocation and opening of the mitochondrial KATP channel in ischemic myocytes.