The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation.

Recent genetic studies have documented a pivotal growth-regulatory role played by the Cullin 7 (CUL7) E3 ubiquitin ligase complex containing the Fbw8-substrate-targeting subunit, Skp1, and the ROC1 RING finger protein. In this report, we identified insulin receptor substrate 1 (IRS-1), a critical mediator of the insulin/insulin-like growth factor 1 signaling, as a proteolytic target of the CUL7 E3 ligase in a manner that depends on mammalian target of rapamycin and the p70 S6 kinase activities. Interestingly, while embryonic fibroblasts of Cul7-/- mice were found to accumulate IRS-1 and exhibit increased activation of IRS-1's downstream Akt and MEK/ERK pathways, these null cells grew poorly and displayed phenotypes reminiscent of those associated with oncogene-induced senescence. Taken together, our findings demonstrate a key role for the CUL7 E3 in targeting IRS-1 for degradation, a process that may contribute to the regulation of cellular senescence.

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.

Cardiomyocyte cell cycle activation ameliorates fibrosis in the atrium.

MHC-TGFcys33ser transgenic mice have elevated levels of active transforming growth factor (TGF)-beta1 in the myocardium. Previous studies have shown that these animals develop atrial, but not ventricular, fibrosis. Here we show that atrial fibrosis was accompanied with cardiomyocyte apoptosis. Although similar levels of cardiomyocyte apoptosis were present in the right and left atria of MHC-TGFcys33ser hearts, the extent of fibrosis was more pronounced in the right atrium. Thus, additional factors influence the degree of atrial fibrosis in this model. Tritiated thymidine incorporation studies revealed cardiomyocyte cell cycle activity in left atrial cardiomyocytes, but not in right atrial cardiomyocytes. These observations suggested that cardiomyocyte cell cycle activation ameliorated the severity of atrial fibrosis. To directly test this hypothesis, MHC-TGFcys33ser mice were crossed with MHC-cycD2 mice (which have constitutive cardiomyocyte cell cycle activity in the right atrium). Mice inheriting both transgenes exhibited right atrial cardiomyocyte cell cycle activity and a concomitant reduction in the severity of right atrial fibrosis, despite the presence of a similar level of cardiomyocyte apoptosis as was observed in mice inheriting the MHC-TGFcys33ser transgene alone. These data support the notion that cardiomyocyte cell cycle induction can antagonize fibrosis in the myocardium.

Targeted expression of cyclin D2 results in cardiomyocyte DNA synthesis and infarct regression in transgenic mice.

Restriction point transit and commitment to a new round of cell division is regulated by the activity of cyclin-dependent kinase 4 and its obligate activating partners, the D-type cyclins. In this study, we examined the ability of D-type cyclins to promote cardiomyocyte cell cycle activity. Adult transgenic mice expressing cyclin D1, D2, or D3 under the regulation of the alpha cardiac myosin heavy chain promoter exhibited high rates of cardiomyocyte DNA synthesis under baseline conditions. Cardiac injury in mice expressing cyclin D1 or D3 resulted in cytoplasmic cyclin D accumulation, with a concomitant reduction in the level of cardiomyocyte DNA synthesis. In contrast, cardiac injury in mice expressing cyclin D2 did not alter subcellular cyclin localization. Consequently, cardiomyocyte cell cycle activity persisted in injured hearts expressing cyclin D2, ultimately resulting in infarct regression. These data suggested that modulation of D-type cyclins could be exploited to promote regenerative growth in injured hearts.

Expression of mutant p193 and p53 permits cardiomyocyte cell cycle reentry after myocardial infarction in transgenic mice.

Previous studies have demonstrated that expression of p193 and p53 mutants with dominant-interfering activities renders embryonic stem cell-derived cardiomyocytes responsive to the growth promoting activities of the E1A viral oncoproteins. In this study, the effects of p53 and p193 antagonization on cardiomyocyte cell cycle activity in normal and infarcted hearts were examined. Transgenic mice expressing the p193 and/or the p53 dominant-interfering mutants in the heart were generated. Transgene expression had no effect on cardiomyocyte cell cycle activity in uninjured adult hearts. In contrast expression of either transgene resulted in a marked induction of cardiomyocyte cell cycle activity at the infarct border zone at 4 weeks after permanent coronary artery occlusion. Expression of the p193 dominant-interfering mutant was also associated with an induction of cardiomyocyte DNA synthesis in the interventricular septa of infarcted hearts. A concomitant and marked reduction in hypertrophic cardiomyocyte growth was observed in the septa of hearts expressing the p193 dominant-interfering transgene, suggesting that cell cycle activation might partially counteract the adverse ventricular remodeling that occurs after infarction. Collectively these data suggest that antagonization of p193 and p53 activity relaxes the otherwise stringent regulation of cardiomyocyte cell cycle reentry in the injured adult heart.

Physiological coupling of donor and host cardiomyocytes after cellular transplantation.

Cellular transplantation has emerged as a potential approach to treat diseased hearts. Although cell transplantation can affect global heart function, it is not known if this results directly via functional integration of donor myocytes or indirectly via enhanced revascularization and/or altered postinjury remodeling. To determine the degree to which donor cardiomyocytes are able to functionally integrate with the host myocardium, fetal transgenic cardiomyocytes expressing enhanced green fluorescent protein were transplanted into the hearts of nontransgenic adult mice. Two-photon molecular excitation laser scanning microscopy was then used to simultaneously image cellular calcium transients in donor and host cells within the intact recipient hearts. Calcium transients in the donor cardiomyocytes were synchronous with and had kinetics indistinguishable from those of neighboring host cardiomyocytes. These results strongly suggest that donor cardiomyocytes functionally couple with host cardiomyocytes and support the notion that transplanted cardiomyocytes can form a functional syncytium with the host myocardium.

Increased vulnerability to atrial fibrillation in transgenic mice with selective atrial fibrosis caused by overexpression of TGF-beta1.

Studies on patients and large animal models suggest the importance of atrial fibrosis in the development of atrial fibrillation (AF). To investigate whether increased fibrosis is sufficient to produce a substrate for AF, we have studied cardiac electrophysiology (EP) and inducibility of atrial arrhythmias in MHC-TGFcys33ser transgenic mice (Tx), which have increased fibrosis in the atrium but not in the ventricles. In anesthetized mice, wild-type (Wt) and Tx did not show significant differences in surface ECG parameters. With transesophageal atrial pacing, no significant differences were observed in EP parameters, except for a significant decrease in corrected sinus node recovery time in Tx mice. Burst pacing induced AF in 14 of 29 Tx mice, whereas AF was not induced in Wt littermates (P<0.01). In Langendorff perfused hearts, atrial conduction was studied using a 16-electrode array. Epicardial conduction velocity was significantly decreased in the Tx RA compared with the Wt RA. In the Tx LA, conduction velocity was not significantly different from Wt, but conduction was more heterogeneous. Action potential characteristics recorded with intracellular microelectrodes did not reveal differences between Wt and Tx mice in either atrium. Thus, in this transgenic mouse model, selective atrial fibrosis is sufficient to increase AF inducibility.