SUMMARY In advanced atherosclerosis, macrophage apoptosis coupled with defective phagocytic clearance of the apoptotic cells (efferocytosis) promotes plaque necrosis, which precipitates acute atherothrombotic cardiovascular events. Oxidative and endoplasmic reticulum (ER) stress in macrophages are important causes of advanced lesional macrophage apoptosis. We now show that pro-apoptotic oxidative/ER stress inducers trigger another stress reaction in macrophages, autophagy. Inhibition of autophagy by silencing ATG5 or other autophagy mediators enhances apoptosis and NADPH oxidase-mediated oxidative stress, while at the same time rendering the apoptotic cells less well recognized by efferocytes. Most importantly, macrophage ATG5 deficiency in fat-fed Ldlr−/− mice increases apoptosis and oxidative stress in advanced lesional macrophages, promotes plaque necrosis, and worsens lesional efferocytosis. These findings reveal a protective process in oxidatively stressed macrophages relevant to plaque necrosis, suggesting a mechanism-based strategy to therapeutically suppress atherosclerosis progression and its clinical sequelae.

Basal autophagy is a crucial mechanism in cellular homeostasis, underlying both normal cellular recycling and the clearance of damaged or misfolded proteins, organelles and aggregates. We showed here that enhanced levels of autophagy induced by either autophagic gene overexpression or voluntary exercise ameliorated desmin-related cardiomyopathy (DRC). To increase levels of basal autophagy, we generated an inducible Tg mouse expressing autophagy-related 7 (Atg7), a critical and rate-limiting autophagy protein. Hearts from these mice had enhanced autophagy, but normal morphology and function. We crossed these mice with CryAB R120G mice, a model of DRC in which autophagy is significantly attenuated in the heart, to test the functional significance of autophagy activation in a proteotoxic model of heart failure. Sustained Atg7-induced autophagy in the CryAB R120G hearts decreased interstitial fibrosis, ameliorated ventricular dysfunction, decreased cardiac hypertrophy, reduced intracellular aggregates and prolonged survival. To determine whether different methods of autophagy upregulation have additive or even synergistic benefits, we subjected the autophagy-deficient CryAB R120G mice and the Atg7-crossed CryAB R120G mice to voluntary exercise, which also upregulates autophagy. The entire exercised Atg7-crossed CryAB R120G cohort survived to 7 months. These findings suggest that activating autophagy may be a viable therapeutic strategy for improving cardiac performance under proteotoxic conditions.

Rationale Increasing evidence suggests that misfolded proteins and intracellular aggregates contribute to cardiac disease and heart failure. Several cardiomyopathies, including the αB-crystallin R120G mutation (CryABR120G) model of desmin-related cardiomyopathy, accumulate cytotoxic misfolded proteins in the form of pre-amyloid oligomers (PAOs) and aggresomes. Impaired autophagic function is a potential cause of misfolded protein accumulations, cytoplasmic aggregate loads and cardiac disease. Atg7, a mediator of autophagosomal biogenesis, is a putative regulator of autophagic function. Objective To determine whether autophagic induction by Atg7 is sufficient to reduce misfolded protein and aggregate content in protein misfolding-stressed cardiomyocytes. Methods and Results To define the gain and loss of function effects of Atg7 expression on CryABR120G protein misfolding and aggregates, neonatal rat cardiomyocytes (RNC) were infected with adenoviruses expressing either wild-type CryAB or CryABR120G, and co-infected with Atg7 adenovirus or with Atg7 silencing siRNAs to produce gain- or loss-of Atg7 function. Atg7 overexpression effectively induced basal autophagy with no detrimental effects on cell survival, suggesting that Atg7 can activate autophagy with no apparent cytotoxic effects. Autophagic flux assays on CryABR120G expressing cardiomyocytes revealed reduced autophagic function, likely contributing to the failure of misfolded proteins and aggregates to be cleared. Co-expression of Atg7 and CryABR120G significantly reduced PAO staining, aggregate content and cardiomyocyte cytotoxicity. Conversely, Atg7 silencing in the CryABR120G background significantly inhibited the already reduced rate of autophagy and increased CryABR120G aggregate content and cytotoxicity. Conclusions Atg7 induces basal autophagy, rescues the CryABR120G autophagic deficiency, and attenuates the accumulation of misfolded proteins and aggregates in cardiomyocytes.

Background-To determine whether soluble preamyloid oligomers (PAOs) are toxic when expressed internally in the cardiomyocyte, we tested the hypothesis that cardiomyocyte-restricted expression and accumulation of a known PAO is cytotoxic and sufficient to cause heart failure. Methods and Results-Intracellular PAOs, the entities believed to cause toxicity in many neurodegenerative diseases, have been observed in cardiomyocytes derived from mouse and human heart failure samples. Long (Ͼ50) polyglutamine (PQ) repeats form PAOs and cause neurotoxicity in Huntington disease and other neurodegenerative diseases, whereas shorter PQ peptides are benign. We created transgenic mice in which cardiomyocyte-autonomous expression of an 83 residue-long PQ repeat (PQ83) or a non-amyloid-forming peptide of 19 PQ repeats (PQ19) as a nonpathological control was expressed. A PQ83 line with relatively low levels of expression was generated, along with a PQ19 line that expressed Ϸ9-fold the levels observed in the PQ83 line. Hearts expressing PQ83 exhibited reduced cardiac function and dilation by 5 months, and all mice died by 8 months, whereas PQ19 mice had normal cardiac function, morphology, and life span. PQ83 protein accumulated within aggresomes with PAO-specific staining. The PQ83 hearts showed increased autophagosomal and lysosomal content but also showed markers of necrotic death, including inflammatory cell infiltration and increased sarcolemmal permeability. Conclusions-The data confirm the hypothesis that expression of an exogenous PAO-forming peptide is toxic to cardiomyocytes and is sufficient to cause cardiomyocyte loss and heart failure in a murine model.

The differences in gene expression among the fiber types of skeletal muscle have long fascinated scientists, but for the most part, previous experiments have only reported differences of one or two genes at a time. The evolving technology of global mRNA expression analysis was employed to determine the potential differential expression of approximately 3,000 mRNAs between the white quad (white muscle) and the red soleus muscle (mixed red muscle) of female ICR mice (30-35 g). Microarray analysis identified 49 mRNA sequences that were differentially expressed between white and mixed red skeletal muscle, including newly identified differential expressions between muscle types. For example, the current findings increase the number of known, differentially expressed mRNAs for transcription factors/coregulators by nine and signaling proteins by three. The expanding knowledge of the diversity of mRNA expression between white and mixed red muscle suggests that there could be quite a complex regulation of phenotype between muscles of different fiber types.