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.

Genetic ablation of Cullin-RING E3 ubiquitin ligase 7 restrains pressure overload-induced myocardial fibrosis.

Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.

Cardiomyocyte cell cycle activation improves cardiac function after myocardial infarction.

AIMS: Cardiomyocyte loss is a major contributor to the decreased cardiac function observed in diseased hearts. Previous studies have shown that cardiomyocyte-restricted cyclin D2 expression resulted in sustained cell cycle activity following myocardial injury in transgenic (MHC-cycD2) mice. Here, we investigated the effects of this cell cycle activation on cardiac function following myocardial infarction (MI). METHODS AND RESULTS: MI was induced in transgenic and non-transgenic mice by left coronary artery occlusion. At 7, 60, and 180 days after MI, left ventricular pressure-volume measurements were recorded and histological analysis was performed. MI had a similar adverse effect on cardiac function in transgenic and non-transgenic mice at 7 days post-injury. No improvement in cardiac function was observed in non-transgenic mice at 60 and 180 days post-MI. In contrast, the transgenic animals exhibited a progressive and marked increase in cardiac function at subsequent time points. Improved cardiac function in the transgenic mice at 60 and 180 days post-MI correlated positively with the presence of newly formed myocardial tissue which was not apparent at 7 days post-MI. Intracellular calcium transient imaging indicated that cardiomyocytes present in the newly formed myocardium participated in a functional syncytium with the remote myocardium. CONCLUSION: These findings indicate that cardiomyocyte cell cycle activation leads to improvement of cardiac function and morphology following MI and may represent an important clinical strategy to promote myocardial regeneration.

Expression of a mutant p193/CUL7 molecule confers resistance to MG132- and etoposide-induced apoptosis independent of p53 or Parc binding.

p193/CUL7 is an E3 ubiquitin ligase initially identified as an SV40 Large T Antigen binding protein. Expression of a dominant interfering variant of mouse p193/CUL7 (designated 1152stop) conferred resistance to MG132- and etoposide-induced apoptosis in U2OS cells. Immune precipitation/Western analyses revealed that endogenous p193/CUL7 formed a complex with Parc (a recently identified parkin-like ubiquitin ligase) and p53. Apoptosis resistance did not result from 1152stop-mediated disruption of the endogenous p193/CUL7 binding partners. Moreover, 1152stop molecule did not directly bind to endogenous p193/CUL7, Parc or p53. These data suggested a role for p193/CUL7 in the regulation of apoptosis independently of p53 and Parc activity.

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.

Spontaneous and evoked intracellular calcium transients in donor-derived myocytes following intracardiac myoblast transplantation.

Skeletal myoblast transplantation is a potential treatment for congestive heart failure. To study the functional activity of both donor and host myocytes following transplantation, skeletal myoblasts expressing an enhanced green fluorescent protein (EGFP) transgene were transplanted into hearts of nontransgenic recipients, and changes in intracellular calcium concentration ([Ca2+]i) were monitored in donor and host cells. While the vast majority of donor-derived myocytes were observed to be functionally isolated from the host myocardium, a small population of donor myocytes exhibited action potential-induced calcium transients in synchrony with adjacent host cardiomyocytes. In many cases, the durations of these [Ca2+]i transients were heterogeneous compared with those in neighboring host cardiomyocytes. In other studies, EGFP-expressing donor myoblasts were transplanted into the hearts of adult transgenic recipient mice expressing a cardiomyocyte-restricted beta-gal reporter gene. A small population of myocytes was observed to express both reporter transgenes, indicating that the transplanted myoblasts fused with host cardiomyocytes at a very low frequency. These cells also expressed connexin43, a component of gap junctions. Thus engraftment of skeletal myoblasts generated spatial heterogeneity of [Ca2+]i signaling at the myocardial/skeletal muscle interface, most likely as a consequence of fusion events between donor myoblasts and host cardiomyocytes.

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.