Even though CARP was first identified as an exquisitely sensitive target of doxorubicin 
, studies examining the relationship between CARP and doxorubicin cardiotoxicity have been scant. In the present work we show that doxorubicin depletes both GATA4 and CARP levels in cultured cardiomyocytes, and that GATA4 overexpression (but not CARP overexpression) was able to attenuate doxorubicin-induced sarcomere disarray. We demonstrate that GATA4 directly regulates CARP and that the protective effect of GATA4 was mediated in part by modulating CARP expression and downstream sarcomere genes. These results are the first to identify CARP as a mediator for GATA4 in a signaling axis that converges to control sarcomere gene expression and maintain organized sarcomeres.
Doxorubicin induces sarcomere disarray and a rapid decrease in CARP protein levels in both ARVM and NRVM, with the phenotype appearing to be more severe in NRVM. CARP is believed to be a “hot spot”, titin-based, mechanosensory unit as it interacts with the elastic N2A domain of titin (for reviews see 
). Consistent with our previous report, doxorubicin induced titin degradation in ARVMs and co-treatment with a calpain inhibitor restored titin levels and preserved myofibrillar structure 
. However, calpain inhibition did not preserve CARP levels suggesting that 1) CARP is not a calpain substrate and 2) the susceptibility of CARP to doxorubicin is independent of a preserved titin and/or sarcomere structure. The latter might be explained by impaired CARP binding to N2A titin, perhaps due to post-translational modifications, leading to enhanced degradation of free CARP by some other proteolytic mechanism. However, in this study ARVMs treated with cycloheximide in the presence or absence of doxorubicin showed no difference in the rate of CARP loss, thus arguing against accelerated CARP degradation; instead we were able to demonstrate that doxorubicin induced transcriptional inhibition of CARP. Support for this comes from a previous study showing doxorubicin-induced suppression of CARP transcription via activation of a H7-sensitive serine/threonine kinase pathway 
Neonatal cardiomyocytes subjected to stretch accumulate CARP in the sarcomeric I-band as well as in the nucleus, suggesting that CARP might couple mechanical strain to muscle gene transcription 
. Following doxorubicin exposure, we also observed a transient sarcomeric to nuclear translocation of CARP. We speculate that in response to doxorubicin-induced mechanical perturbation (titin degradation) sarcomeric CARP localizes to the nucleus to modulate myofilament gene transcription; this gene transcription ultimately ceases as total CARP levels are eventually depleted with doxorubicin. It is unclear how CARP regulates cardiac gene expression, but CARP is known to be a transcriptional co-factor and has been shown to interact in vitro
with multiple transcription factors involved in cardiac gene expression, including YB-1, HAND2, and HEY1 
The cardiac transcription factor GATA4 plays a pivotal role in cardiomyocyte hypertrophy and survival and it has been implicated in sarcomere gene transcription and regulation of the sarcomere assembly process 
. These diverse processes are likely mediated by different downstream effectors, which remain poorly defined. GATA4 is known to be sensitive to doxorubicin 
. Here we show that selective siRNA knockdown of GATA4 suppressed CARP promoter activity, depleted CARP protein levels, and induced extensive cardiomyocyte sarcomere disarray. These findings suggest that doxorubicin-induced depletion of GATA4 is directly responsible for loss of CARP, and they implicate CARP as a downstream mediator of GATA4 in regulating sarcomere maintenance. Overexpression of GATA4 enhanced CARP promoter activity in HEK293 cells whereas GATA4 siRNA knockdown in cardiomyocytes resulted in suppression of CARP promoter activity, confirming that GATA4 directly regulates CARP. CARP siRNA knockdown induced marked cardiomyocyte sarcomere disarray as seen with GATA4 siRNA, and either CARP or GATA4 siRNA resulted in significant attenuation in promoter activity of sarcomere genes, titin and α-actin. Overexpression of CARP failed to rescue the doxorubicin-induced sarcomere disarray phenotype, which was not unexpected as the concomitant loss of GATA4 is predicted to inhibit other downstream pathways distinct from sarcomeric organization such as survival pathways 
. Furthermore, CARP is hypothesized to be a mechanosensor linking changes in titin mechanical strain to gene transcription, and it is unclear how overexpression of ectopic CARP might affect this endogenous mechanosensing or transcriptional activity of CARP. It is interesting that CARP promoter activity was inversely related to CARP expression, suggesting a negative feedback mechanism to strictly control CARP levels in cardiomyocytes. As previously noted, CARP is a cofactor for multiple cardiac transcription factors and stringent regulation of CARP is perhaps necessary to limit an exaggerated hypertrophic response. Future studies are needed to examine the role of CARP in cardiac hypertrophy.
Overexpression of GATA4, on the other hand, markedly attenuated doxorubicin-induced cardiomyocyte sarcomere disarray. This finding is consistent with previous studies which showed that GATA4 overexpression protects against cardiomyocyte apoptosis and autophagy due to doxorubicin toxicity 
. These previous studies indicated that GATA4 upregulates antiapoptotic factors Bcl-X and Bcl2, and Bcl2 in turn suppresses autophagy-related genes 
. Interestingly, GATA4 overexpression modestly, but significantly, increased CARP levels in doxorubicin treated cardiomyocytes. Our finding that knockdown of this modest increase in CARP completely abrogated the rescue of GATA4 overexpression in doxorubicin treated cells suggest that CARP and GATA4 are both essential in maintaining organized sarcomeres. Our finding that CARP plays a central role in sarcomere maintenance in cultured cardiomyocytes, appears to contradict a study by Barash et al. which showed a relatively mild skeletal muscle phenotype in a triple KO mouse model of the MARP genes (including CARP), with no overt cardiac phenotype reported 
. One possible explanation is that other titin-based mechanosensors can compensate for the lack of CARP in vivo
, e.g the MLP/telethonin/N-titin, MURF2/titin kinase, or FHL1/N2B-titin complexes 
. The possibility that CARP is dispensable for basal in vivo
cardiac function but is important under conditions of stress, such as pressure overload or myocardial infarction, requires further investigation. We also cannot rule out the possibility that when cultured in vitro
, cardiomyocytes remodel and adapt to the rigid two-dimensional environment, where CARP becomes absolutely essential for sarcomere integrity.
GATA4 and the cardiac-enriched transcription factor Nkx2.5 are known mutual co-activators 
, and a previous study has shown that Nkx2.5 and GATA4 cooperatively regulate CARP expression 
. The GATA4/Nkx2.5 interaction has been shown to mediate a mechanical stretch-activated hypertrophic program in cardiomyocytes that enhances sarcomere assembly and organization 
. We speculate that Nkx2.5, GATA4, and CARP all converge on a final common signaling pathway to regulate sarcomere gene transcription and sarcomere maintenance, and that loss of any one of these factors results in cardiomyocyte sarcomere disarray. We further speculate that cardiomyocyte mechanical stretch targets this same Nkx2.5/GATA4/CARP signaling pathway to induce cardiomyocyte hypertrophy and enhance sarcomere organization and assembly. A recent study showed that Ankrd2, another member of the MARP family of proteins enriched in skeletal muscle, interacts with transcriptional regulators and structural and signaling proteins to affect a multitude of pathways including myogenesis, gene expression, as well as intra- and intercellular signaling 
. We anticipate that future studies on CARP will uncover novel signaling pathways and processes with important regulatory functions in the heart.
The mechanisms of anthracycline cardiotoxicity are diverse and include 1) oxidative stress and membrane lipid damage, 2) calcium overload, and 3) inhibition of protein transcription and translation (reviewed in 
). These conditions collectively lead to myofibrillar disarray, where acceleration of myofilament protein degradation and simultaneous repression of muscle gene expression leads to a net negative balance of sarcomeric proteins. In this paradigm, the downregulation of GATA4 and CARP contributes in part to this phenotype by preventing synthesis of sarcomere proteins and sarcomere assembly (). The hypothesis that CARP/N2A-titin make up a stretch-sensing unit that intersects with Nkx2.5/GATA4 signaling is particularly attractive as a mechanism for cardiomyocytes to rapidly adjust sarcomere homeostasis to the changing physiological demands of the heart.
Cardiac sarcomere structure is maintained by a balance between sarcomere protein synthesis and degradation.