Background—: The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Methods and Results—: Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1 . Beclin 1 ^+/− mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Conclusions—: Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity.

Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member–encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-β–activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1–MKK3/6–p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38β and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38β agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging.

Rho-associated coiled-coil protein kinase 1 (ROCK-1) is a direct cleavage substrate of activated caspase-3, which is associated with heart failure. In the course of human heart failure, we found marked cleavage of ROCK-1 resulting in a 130-kDa subspecies, which was absent in normal hearts and in an equivalent cohort of patients with left ventricular assist devices. Murine cardiomyocytes treated with doxorubicin led to enhanced ROCK-1 cleavage and apoptosis, all of which was blocked by a caspase-3 inhibitor. In addition, a bitransgenic mouse model of severe cardiomyopathy, which overexpresses Gq protein and hematopoietic progenitor kinase-/germinal center kinase-like kinase, revealed the robust accumulation of the 130-kDa ROCK-1 cleaved fragment. This constitutively active ROCK-1 subspecies, when expressed in cardiomyocytes, led to caspase-3 activation, indicating a positive feed-forward regulatory loop. ROCK-1-dependent caspase-3 activation was coupled with the activation of PTEN and the subsequent inhibition of protein kinase B (Akt) activity, all of which was attenuated by siRNA directed against ROCK-1 expression. Similarly, ROCK-1-null mice (Rock-1^−/−) showed a marked reduction in myocyte apoptosis associated with pressure overload. These data suggest an obligatory role for ROCK-1 cleavage in promoting apoptotic signals in myocardial hypertrophy and/or failure.

The protein kinase TAK1 mediates the activation of NF-κB in response to stimulation by proinflammatory cytokines and microbial pathogens in the innate immunity pathways. However, the physiological function of TAK1 in the adaptive immunity pathways is unclear. By engineering mice lacking TAK1 in T cells, here, we show that TAK1 is essential for thymocyte development and activationin vivo. Deletion of TAK1 prevented the maturation of single-positive thymocytes displaying CD4 or CD8, leading to reduction of T cells in the peripheral tissues. Thymocytes lacking TAK1 failed to activate NF-κB and JNK and were prone to apoptosis upon stimulation. Our results provide the genetic evidence that TAK1 is required for the activation of NF-κB in thymocytes and suggest that TAK1 plays a central role in both innate and adaptive immunity.