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.

Ventricular myocyte hypertrophy is an important compensatory growth response to pressure overload. However, pathophysiological cardiac hypertrophy is accompanied by reactive fibrosis and remodeling. The Rho kinase family, consisting of ROCK1 and ROCK2, has been implicated in cardiac hypertrophy and ventricular remodeling. However, these previous studies relied heavily on pharmacological inhibitors, and not on gene deletion. Here we used ROCK1 knockout (ROCK1 ^−/− ) mice to investigate role of ROCK1 in the development of ventricular remodeling induced by transverse aortic banding. We observed that ROCK1 deletion did not impair compensatory hypertrophic response induced by pressure overload. However, ROCK1 ^−/− mice exhibited reduced perivascular and interstitial fibrosis, which was observed at 3 wk but not at 1 wk after the banding. The reduced fibrosis in the myocardium of ROCK1 ^−/− mice was closely associated with reduced expression of a variety of extracellular matrix (ECM) proteins and fibrogenic cytokines such as TGFβ2 and connective tissue growth factor. This inhibitory effect of ROCK1 deletion on pathophysiological induction of fibrogenic cytokines was further confirmed in the myocardium of transgenic mice with cardiomyocyte‐specific overexpression of Gαq. Thus, these results indicate that ROCK1 contributes to the development of cardiac fibrosis and induction of fibrogenic cytokines in cardiomyocytes in response to pathological stimuli. Zhang, Y.‐M., Bo, J., Taffet, G. E., Chang, J., Shi, J., Reddy, A. K., Michael, L. H., Schneider, M. D., Entman, M. L., Schwartz, R. J., Wei, L. Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis. FASEB J. 20, 916–925 (2006)

Heat shock proteins (Hsps) are well appreciated as intrinsic protectors of cardiomyocytes against numerous stresses. Recent studies have indicated that Hsp20 (HspB6), a small heat shock protein, was increased in blood from cardiomyopathic hamsters. However, the exact source of the increased circulating Hsp20 and its potential role remain obscure. In this study, we observed that the circulating Hsp20 was increased in a transgenic mouse model with cardiac-specific overexpression of Hsp20, compared with wild-type mice, suggesting its origin from cardiomyocytes. Consistently, culture media harvested from Hsp20-overexpressing cardiomyocytes by Ad.Hsp20 infection contained an increased amount of Hsp20, compared to control media. Furthermore, we identified that Hsp20 was secreted through exosomes, independent of the endoplasmic reticulum-Golgi pathway. To investigate whether extracellular Hsp20 promotes angiogenesis, we treated human umbilical vein endothelial cells (HUVECs) with recombinant human Hsp20 protein, and observed that Hsp20 dose-dependently promoted HUVEC proliferation, migration and tube formation. Moreover, a protein binding assay and immunostaining revealed an interaction between Hsp20 and VEGFR2. Accordingly, stimulatory effects of Hsp20 on HUVECs were blocked by a VEGFR2 neutralizing antibody and CBO-P11 (a VEGFR inhibitor). These in vitro data are consistent with the in vivo findings that capillary density was significantly enhanced in Hsp20-overexpressing hearts, compared to non-transgenic hearts. Collectively, our findings demonstrate that Hsp20 serves as a novel cardiokine in regulating myocardial angiogenesis through activation of the VEGFR signaling cascade.

Cancer development involves the co-evolution of cancer cells and their surrounding microenvironment, yet the dynamics of this interaction within the physical architecture remains poorly understood. Here, we present a spatial transcriptomic map at single-cell resolution, encompassing 127 multi-stage fields of view from 43 patients, to chart the evolutionary trajectories of human esophageal squamous cell carcinoma (ESCC). By analyzing 6.4 million cells, we reveal that ESCC progression is driven by a proliferative epithelial cell subpopulation that acquires dedifferentiated and invasive characteristics. At the late precancerous stage, these cells disrupt the epithelial-stromal interface and recruit normal fibroblasts via JAG1-NOTCH1 signaling, transforming them into cancer-associated fibroblasts (CAFs). This interaction leads to the formation of a "CAF-Epi" (CAF and epithelial cell) niche at the tumor edge that shields the tumor from immune surveillance. The CAF-Epi niche formation is a key indicator of progression in ESCC and other squamous cell carcinomas and patient outcomes.

Background: The nuclear receptor Rev-erbα/β, a key component of the circadian clock, emerges as a drug target for heart diseases, but the function of cardiac Rev-erb has not been studied in vivo. Circadian disruption is implicated in heart diseases, but it is unknown whether cardiac molecular clock dysfunction is associated with the progression of any naturally occurring human heart diseases. Obesity paradox refers to the seemingly protective role of obesity for heart failure, but the mechanism is unclear. Methods: We generated mouse lines with cardiac-specific Rev-erbα/β knockout (KO), characterized cardiac phenotype, conducted multi-omics (RNA-seq, ChIP-seq, proteomics, and metabolomics) analyses, and performed dietary and pharmacologic rescue experiments to assess the time-of-the-day effects. We compared the temporal pattern of cardiac clock gene expression with the cardiac dilation severity in failing human hearts. Results: KO mice display progressive dilated cardiomyopathy (DCM) and lethal heart failure. Inducible ablation of Rev-erbα/β in adult hearts causes similar phenotypes. Impaired fatty acid oxidation in the KO myocardium, particularly in the light cycle, precedes contractile dysfunctions with a reciprocal overreliance on carbohydrate utilization, particularly in the dark cycle. Increasing dietary lipids or sugars supply in the dark cycle does not affect cardiac dysfunctions in KO mice. However, obesity coupled with systemic insulin resistance paradoxically ameliorates cardiac dysfunctions in KO mice, associated with rescued expression of lipid oxidation genes only in the light cycle in phase with increased fatty acids availability from adipose lipolysis. Inhibition of glycolysis in the light cycle and lipid oxidation in the dark cycle, but not vice versa, ameliorates cardiac dysfunctions in KO mice. Altered temporal patterns of cardiac Rev-erb gene expression correlate with the cardiac dilation severity in human hearts with DCM. Conclusions: The study delineates temporal coordination between clock-mediated anticipation and nutrient-induced response in myocardial metabolism at multi-omics levels. The obesity paradox is attributable to increased cardiac lipids supply from adipose lipolysis in the fasting cycle due to systemic insulin resistance and adiposity. Cardiac molecular chronotypes may be involved in human DCM. Myocardial bioenergetics downstream of Rev-erb may be a chronotherapy target in treating heart failure and DCM.