Several cardiac troponin I (cTnI) mutations are associated with restrictive cardiomyopathy (RCM) in humans. We have created transgenic mice (cTnI193His mice) that express the corresponding human RCM R192H mutation. Phenotype of this RCM animal model includes restrictive ventricles, biatrial enlargement and sudden cardiac death, which are similar to those observed in RCM patients carrying the same cTnI mutation. In the present study, we modified the overall cTnI in cardiac muscle by crossing cTnI193His mice with transgenic mice expressing an N-terminal truncated cTnI (cTnI-ND) that enhances relaxation. Protein analyses determined that wild type cTnI was replaced by cTnI-ND in the heart of double transgenic mice (Double TG), which express only cTnI-ND and cTnI R193H in cardiac myocytes. The presence of cTnI-ND effectively rescued the lethal phenotype of RCM mice by reducing the mortality rate. Cardiac function was significantly improved in Double TG mice when measured by echocardiography. The hypersensitivity to Ca2+ and the prolonged relaxation of RCM cTnI193His cardiac myocytes were completely reversed by the presence of cTnI-ND in RCM hearts. The results demonstrate that myofibril hypersensitivity to Ca2+ is a key mechanism that causes impaired relaxation in RCM cTnI mutant hearts and Ca2+ desensitization by cTnI-ND can correct diastolic dysfunction and rescue the RCM phenotypes, suggesting that Ca2+ desensitization in myofibrils is a therapeutic option for treatment of diastolic dysfunction without interventions directed at the systemic β-adrenergic-PKA pathways.

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Objectives To explore the dose-dependent diastolic dysfunction and the mechanisms of heart failure and early death in transgenic (TG) mice modeling human restrictive cardiomyopathy (RCM). Background The first RCM mouse model (cTnI193His mice) carrying cardiac troponin I (cTnI) R193H mutation (mouse cTnI R193H equals to human cTnI R192H) was generated several years ago in our laboratory. The RCM mice manifested a phenotype similar to that observed in RCM patients carrying the same cTnI mutation, i.e. enlarged atria and restricted ventricles. However, the causes of heart failure and early death observed in RCM mice remain unclear. Methods In this study, we have produced RCM TG mice (cTnI193His-L, cTnI193His-M and cTnI193His-H) that express various levels of mutant cTnI in the heart. Histological examination and echocardiography were performed in these mice to monitor the time course of the disease development and heart failure. Results Our data demonstrate that cTnI mutation-caused diastolic dysfunction is dose-dependent. The key mechanism is myofibril hypersensitivity to Ca2+ resulting in an impaired relaxation in the mutant cardiac myocytes. Prolonged relaxation time and delay of Ca2+ decay observed in the mutant cardiac myocytes are correlated with the level of the mutant protein in the heart. Markedly enlarged atria due to the elevated end-diastolic pressure and myocardial ischemia are observed in the heart of the transgenic mice. In the mice with the highest level of the mutant protein, restricted ventricles and systolic dysfunction occurs followed immediately by heart failure and early death. Conclusions Diastolic dysfunction caused by R193H troponin I mutation is specific, showing a dose-dependent pattern. These mouse models are useful tools for study of diastolic dysfunction. Impaired diastole can cause myocardial ischemia and fibrosis formation, resulting in the development of systolic dysfunction and heart failure with early death in the RCM mice with a high level of the mutant protein in the heart.

Abstract. This study was designed to examine the ability of rabbit metaphase II oocyte cytoplasm to support the development of interspecies nuclear transfer embryos reconstructed using donor nuclei from different species. Skin fibroblast cells from a camel and Tibetan antelope were used as donor nuclei. As a first step, we investigated the efficiency of different activation protocols by comparing the parthenogenetic development of rabbit oocytes. The protocol that yielded the highest blastocyst rate was used to activate the reconstructed embryos in nuclear transfer experiments. In addition, the effect of donor cell serum starvation on the development of the reconstructed embryo was also examined. More than half of the karyoplast-cytoplast couplets could be fused, and about one third of the reconstructed embryos were capable of completing first cleavage, regardless of the species of donor nuclei. Some of the cleaving reconstructed embryos were even capable of progressing further and developing to the blastocyst stage (1.4-8.7% for the Tibetan antelope and 0-7.5% for the camel, respectively). Our results suggest that the mechanisms regulating early embryo development may be conserved among mammalian species and some factors existing in rabbit oocyte cytoplasm for somatic nucleus reprogramming and dedifferentiation may not be species-specific. Rabbit oocyte cytoplasm can reprogram donor nuclei regardless of the origin of the nucleus and support in vitro development to an advanced stage. [12]. At the same time, interspecies somatic cell cloned gaur and mouflon [13,14] have been obtained. Interspecies nuclear transfer, which involves transferring a donor cell from one species into a recipient oocyte of another species, is an invaluable tool for studying nucleuscytoplasm interaction and may be an effective way to conserve endangered species whose oocytes are extremely difficult and even impossible to obtain. Moreover, when interspecies nuclear transfer is used for therapeutic research, ethical, legal, and experimental limitations encountered in clinical situations may be avoided. One of the problems of interspecies nuclear transfer is the unavailability of species-specific competent recipient cytoplasm. In particular, it is extremely difficult to get oocytes for