Background Cardiac overload, a major cause of heart failure, induces the expression of the heat shock protein H11 kinase/Hsp22 (Hsp22). Methods and Results To determine the specific function of Hsp22 in that context, a knockout (KO) mouse model of Hsp22 deletion was generated. Although comparable to wild type mice in basal conditions, KO mice exposed to pressure overload developed less hypertrophy, and showed ventricular dilation, impaired contractile function, increased myocyte length and accumulation of interstitial collagen, faster transition into heart failure and increased mortality. Microarrays revealed that hearts from KO mice failed to transactivate genes regulated by the transcription factor STAT3. Accordingly, nuclear STAT3 tyrosine phosphorylation was decreased in KO. Silencing and over-expression experiments in isolated neonatal rat cardiomyocytes showed that Hsp22 activates STAT3 via production of interleukin-6 by the transcription factor NF-κB. In addition to its transcriptional function, STAT3 also translocates to the mitochondria where it increases oxidative phosphorylation. Both mitochondrial STAT3 translocation and respiration were significantly decreased in KO mice as well. Conclusions Hsp22 represents a previously undescribed activator of both nuclear and mitochondrial functions of STAT3, and its deletion in a context of pressure overload in vivo accelerates the transition into heart failure and increases mortality.
In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, α‐sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin–proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21WAF1/CIP1, is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast‐twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21WAF1/CIP1 is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.
A multiprotein complex encompassing a transcription regulator, cardiac ankyrin repeat protein (CARP), and the calpain 3 protease was identified in the N2A elastic region of the giant sarcomeric protein titin. The present study aimed to investigate the function(s) of this complex in the skeletal muscle. We demonstrate that CARP subcellular localization is controlled by the activity of calpain 3: the higher the calpain 3, the more important the sarcomeric retention of CARP. This regulation would occur through cleavage of the N‐terminal end of CARP by the protease. We show that, upon CARP over‐expression, the transcription factor nuclear factor NF‐κB p65 DNA‐binding activity decreases. Taken as a whole, CARP and its regulator calpain 3 appear to occupy a central position in the important cell fate‐governing NF‐κB pathway. Interestingly, the expression of the atrophying protein MURF1, one of NF‐κB main targets, remains unchanged in presence of CARP, suggesting that the pathway encompassing calpain3/CARP/NF‐κB does not play a role in muscle atrophy. With NF‐κB also having anti‐apoptotic effects, the inability of calpain 3 to lower CARP‐driven inhibition of NF‐κB could reduce muscle cell survival, hence partly accounting for the dystrophic pattern observed in limb girdle muscular dystrophy 2A, a pathology resulting from the protease deficiency. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7990388: Titin (uniprotkb:http://www.uniprot.org/uniprot/Q8WZ42) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with CARP (uniprotkb:http://www.uniprot.org/uniprot/Q9CR42) by two hybrid (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0018) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7990374: calpain 3 (uniprotkb:http://www.uniprot.org/uniprot/P20807) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with Titin (uniprotkb:http://www.uniprot.org/uniprot/Q8WZ42) by two hybrid (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0018) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7990342: calpain 3 (uniprotkb:http://www.uniprot.org/uniprot/P20807) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with CARP (uniprotkb:http://www.uniprot.org/uniprot/Q9CR42) by two hybrid (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0018)
H11 kinase/Hsp22 (Hsp22) is a small heat shock protein, which, when overexpressed cardiac specifically in transgenic (TG) mice, induces stable left ventricular (LV) hypertrophy. Hsp22 also increases oxidative phosphorylation and mitochondrial reactive oxygen species (ROS) production, mechanisms mediating LV hypertrophy, senescence and reduced lifespan. Therefore, we investigated whether ROS production mediates LV hypertrophy, senescence and reduced life span in Hsp22 TG mice. Survival curves revealed that TG mice had a 48% reduction in their mean life span compared to wild type (WT) mice. This was associated with a significant increase in senescence markers, such as p16, p19 mRNA levels as well as the percentage of β-galactosidase positive cells and telomerase activity. Oxidized (GSSG)/reduced (GSH) glutathione ratio, an indicator of oxidative stress, and ROS production from 3 major cellular sources was measured in cardiac tissue. Hearts from TG mice exhibited a decrease in GSH/GSSG ratio together with increased ROS production from all sources. To study the role of ROS, mice were treated with the antioxidant Tempol from weaning to their sacrifice. Chronic Tempol treatment abolished oxidative stress and overproduction of ROS, and reduced myocardial hypertrophy and Akt phosphorylation in TG mice. Tempol also significantly extended life span and prevented aging markers in TG mice. Taken together these results show that overexpression of Hsp22 increases oxidative stress responsible for the induction of hypertrophy and senescence and ultimately reduction in life span.
H11 kinase/Hsp22 (H11K) is a small heat shock protein which provides powerful cardioprotection, i.e., reduction by 80% of infarct size expressed as a fraction of area at risk following 45 min coronary occlusion/4 hr reperfusion when over-expressed in a cardiac-specific transgenic (TG) mouse model. The goal of this investigation was to determine if these protective mechanisms would also enhance longevity and be effective in protecting from age-related myocardial dysfunction. Surprisingly, TG mice showed a reduction of 48% in their mean life span as compared to wild type (WT). The mechanism of premature death was most likely cardiac as left ventricular ejection fraction was reduced in one year old TG mice (49% vs 74% in WT, p<0.01), left ventricular cavity was dilated and cardiac hypertrophy was increased by 58%, all compatible with severe dilated cardiomyopathy. At the molecular level, hearts from TG mice showed a significant increase in reactive oxygen species, oxidized/reduced glutathione ratio, NADPH and xanthine oxidase activities, Nox2 expression associated with an increase in p16, p19 mRNA, β-galactosidase positive cells and telomerase activity. Subgroups of WT and TG mice were also treated with the antioxidant tempol (100 mg/kg/d) from weaning to their sacrifice. Chronic tempol treatment abolished oxidative stress, reduced cardiac hypertrophy, extended life span (+31%) and prevented aging markers (p16, p19 mRNA, β-galactosidase positive cells) in TG mice. The marked reduction in infarct size with ischemia/reperfusion noted above, observed in 10-12 weeks old mice, remained unchanged after tempol in TG mice and was not abolished with aging. Thus, this is the first demonstration that the mechanisms mediating reduced longevity such as increased oxidative stress do not reduce cardioprotection in H11K TG mice, and conversely, the mechanisms mediating powerful cardioprotection against ischemic stress are ineffective in maintaining normal cardiac function with aging.
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