生物谷报道：在本周的Cell上的封面文章报道了以华人学者为主的一项重要研究成果，是付向东，陈和平和肖瑞平三位华人的重要发现。我们知道，机体从青年到成年的发育过程中，会发生组织和器官水平的重塑现象，这种重塑现象依赖于外界环境和条件。同样在许多疾病条件下，如心肌肥厚，心力衰竭等条件下，心脏会发生病理性重塑，但心脏的发育和重塑中作用的机理并不是十分清楚。这一研究发现内质网（SR）上一个蛋白ASF/SF2基因在心脏的发育和重塑中可能扮演着中心的角色。采用该基因敲除的小鼠进行研究，发现该基因是通过调节CaMKIIδ，从而调节了心脏的兴奋收缩耦联的过程，该基因缺失会导致心脏兴奋收缩耦联的丧失，从而严重影响了心脏的发育和重塑。
这一研究结果显示了ASP/SF2这一内质网上蛋白可能在心脏发育和重塑中起到关键性作用，它的发现为将来心肌发育和许多心脏疾病提供了新的药物靶点，具有极其重要的意义，同时Cell杂志对该篇文章作了高度的评价，认为：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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