生物谷報道：在本周的Cell上的封面文章報道了以華人學者為主的一項重要研究成果,，是付向東,，陳和平和肖瑞平三位華人的重要發(fā)現。我們知道,，機體從青年到成年的發(fā)育過程中,，會發(fā)生組織和器官水平的重塑現象，這種重塑現象依賴于外界環(huán)境和條件,。同樣在許多疾病條件下,，如心肌肥厚，心力衰竭等條件下,，心臟會發(fā)生病理性重塑,，但心臟的發(fā)育和重塑中作用的機理并不是十分清楚。這一研究發(fā)現內質網（SR）上一個蛋白ASF/SF2基因在心臟的發(fā)育和重塑中可能扮演著中心的角色,。采用該基因敲除的小鼠進行研究,，發(fā)現該基因是通過調節(jié)CaMKIIδ，從而調節(jié)了心臟的興奮收縮耦聯的過程,，該基因缺失會導致心臟興奮收縮耦聯的喪失,，從而嚴重影響了心臟的發(fā)育和重塑。
這一研究結果顯示了ASP/SF2這一內質網上蛋白可能在心臟發(fā)育和重塑中起到關鍵性作用,，它的發(fā)現為將來心肌發(fā)育和許多心臟疾病提供了新的藥物靶點,，具有極其重要的意義，同時Cell雜志對該篇文章作了高度的評價,，認為：Alternative Splicing Regulation Impacts Heart Development,。
Figure 2. Histopathological Analysis of ASF/SF2-Deficient Heart(A) H&E staining of coronal sections of normal and mutant heart from 3- and 7-week-old mice. While the morphology looks identical at 3 weeks, chamber enlargement and thinner posterior wall are evident in the heart from a 7-week-old ASF/SF2 ko mouse in comparison with wt littermate controls.(B) Enlarged sections of H&E staining revealing cardiomyocyte hypertrophy in a 5-week-old ASF/SF2-deficient heart.(C) Trichrome staining showing extensive fibrosis and myofibril disarray in the heart from a mutant mouse 7 weeks after birth.(D) EM analysis of the sarcomere structure in WT and mutant heart from 6-week-old mice. Shortening of the sarcomere and disorganization of the Z-disc (indicated by white arrowheads) are evident in the mutant heart. Magnification, 6610×.
Figure 3. Cardiomyocytes from ASF/SF2 Knockout Mice Display a Hypercontraction Phenotype(A) Transillumination images of single myocytes isolated from 5-week-old wt and ASF/SF2 mutant hearts. In comparison with wt, cellular hypertrophy is evident with the mutant cell displaying ~20% longer diastolic cell length (n = 24–26 cells from three hearts, p < 0.001).(B) Confocal measurement of intracellular Ca2+ transients and contraction. Cells were loaded with fluo-4 and paced electrically at 1.0 Hz. Time and space are displayed on the abscissa and ordinate of the line-scan images, respectively. Right panels show the traces of spatially averaged Ca2+ transients (top) and the corresponding cell shortenings (bottom, downward deflections).(C and D) Statistical analysis of twitch amplitude (TA, percent of diastolic cell length) and peak Ca2+ transient (F/F0, where F0 refers to fluo-4 signal at rest), respectively. N = 24–27 cells from three hearts.(E) Diastolic cytosol Ca2+ level at 1 Hz pacing, indexed by indo-1 fluorescent ratio r410/490. N = 12–14 cells from two hearts.(F) Sarcoplasmic reticulum Ca2+ store. Caffeine (20 mM) was applied to empty the stored Ca2+ into the cytosol. The peak increase of indo-1 fluorescent ratio (Δr410/490) was used as the index of the size of store Ca2+ load. N = 12–14 cells from two hearts. *p < 0.05, **p < 0.01, and ***p < 0.001 KO (filled bars) versus wt (open bars) by Student's t test.
Figure 4. Elevated Ca2+ Leak in ASF/SF2-Deficient Myocytes Measured by Spontaneous Ca2+ Sparks(A) Confocal line-scan, time-lapse images showing spontaneous Ca2+ sparks in single cells isolated from ASF/SF2 KO and wt mice. Measurement was conducted on Fluo-4 loaded cells at rest.(B–D) Comparison of spark frequency (B), amplitude (expressed as F/F0) (C), and full width at half maximum (FWHM) (D) between groups. N = 449–1115 sparks with 21–22 cells from two hearts in each group. ***p < 0.001 KO (filled bars) versus wt (open bars) by Student's t test.
Figure 6. Switch of CaMKIIδ to a Neuronal-Specific Isoform in ASF/SF2-Deficient Heart(A) Schematic illustration of main components involved in excitation-contraction (E-C) coupling in cardiac muscle. Concentrated at the T-tubules are the following: NCX, Na+-Ca2+ exchanger, which plays a minor role in Ca2+ influx and clearance in adult heart; DHPR, dihydropyridine receptor or L-type Ca2+ channel, which is sensitive to membrane depolarization induced by the conduction system; RyR, ryanodine receptor 2, a major Ca2+ release channel in the process of Ca2+ induced-Ca2+ release (CICR). ATP, the SERCA2a ATPase, which is the major channel for Ca2+ reuptake; SR, sarcoplasmic reticulum (note: not to be confused with the SR family of proteins); MF, myofilaments; PLB, phospholamban.(B) Western blotting analysis of some critical components involved in E-C coupling. P-RyR2, phosphorylated RyR2. SERCA2a, sarcoplasmic or endoplasmic reticulum calcium 2 ATPase. PLB, total phospholamban. PLB-Thr17, PLB phosphorylated by CaMKIIδ at threonine 17. PLB-Ser16, PLB phosphorylated by protein kinase A at Serine 16. α-tubulin serves as a loading control.(C) Time course analysis of CaMKIIδ alternative splicing in ASF/SF2-deficient heart. Illustrated in the top are three alternative exons (14, 15, and 16) in the CaMKIIδ gene and the three major isoforms containing exons 15 and 16 (δA), exon 14 (δB), and no alternative exons (δC). Results of RT-PCR analysis show the expression of the neuronal-specific δA isoform in later development in ASF/SF2-deficient heart, which is not the case in SC35-deleted heart from adult mice.(D) Western blotting analysis of CaMKIIδ protein expression in wt and ASF/SF2-deficient heart.(E) Localization of individual CaMKIIδ isoforms. Cardiacmyocytes from the δA transgenic mice were isolated and stained with an anti-HA antibody. A clear striated pattern is observed. This is in contrast to the nuclear staining of the δB isoform and the largely diffused cytoplasmic localization of the δC isoform in isolated myocytes transduced by adenovirus.
Figure 7. The Expression of CaMKIIδA in Transgenic Mice Phenocopies the Ca2+ Handling Defects in ASF/SF2-Deficient Myocytes(A) Diagram of the transgenic construct. αMHC, mouse α myosin heavy chain promoter. HA, hemagglutinin tag. SV40, polyadenylation signal of simian virus 40.(B) Genotyping of transgenic mice (TG).(C) Measurement of major Ca2+ handling parameters in transgenic mice. Upper panels show typical fluo-4 and confocal images of CaMKIIδA TG and wt littermate cells at 1.0 Hz pacing. Lower panels show the corresponding traces of spatially averaged Ca2+ transients (top) and the corresponding cell shortenings (bottom, downward deflections).(D–F) Statistic data of twitch amplitude (TA) (D), peak Ca2+ transient (F/F0) (E), and caffeine-elicited Ca2+ transients after 1 Hz pacing (indo-1 fluorescent ratio Δr410/490) (F). N = 30–40 cells from three hearts. *p < 0.05 and **p < 0.01 TG (filled bars) versus wt (open bars) by Student's t test.
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