Exercise-induced CITED4 expression is required for regional helicity remodeling of cardiac microstructural tissues

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In this study, the spatial distribution and upregulation of the physiological growth marker CITED4 was determined in exercised mouse heart tissue using RNA-FISH and compared to microstructural tissue biomarkers based on DT-MRI. . The trend for a total increase in the CITED4/DAPI ratio in exercised heart tissue was consistent with previous reports using qPCR2,7,8. However, RNA-FISH also allowed for more focused spatial analysis which revealed increased CITED4/DAPI ratios mainly in the lateral wall. Additionally, ex vivo anatomical cardiac MRI of sedentary and exercised hearts confirmed the increase in left ventricular mass by exercise.1.2 and myocardial microstructural characterization revealed the microstructural alterations characterized by helicity in the left ventricle of exercised mice. The direct spatial correlation of these microstructural tissues and molecular changes could provide additional insight into how molecular expression may manifest in gross anatomical and physiological remodeling. To our knowledge, this is the first study that investigates the interplay between regional gene expression and microstructural tissue remodeling, providing insight into how tissue-level remodeling can arise from molecular mechanisms.

DT-MRI is a powerful tool for determining small-scale fiber orientation changes associated with disease pathology30.31. In this study, DT-MRI of exercised mice revealed microstructural changes similar to previous findings in mouse models of myocardial infarction treated with cell therapy injections.31, which included significant changes in myocardial fiber helicity. However, in this study, these changes in microstructural helicity are directly related to exercise-induced structural remodeling, which results in improved outcomes after heart injury and disease.30. These exercise-induced helicity changes shown in this study further demonstrate the structural cardiac remodeling benefits of exercise, and in conjunction with previous work30, help demonstrate exercise-induced structural changes that may be cardioprotective. Since DT-MRI can be performed in vivo and non-invasively19,20,21,22,23potentially these CITED4-induced myocardial microstructural changes could be explored to see if they are cardioprotective in a lifesaving preclinical study.

Conventional qPCR has been commonly used as a standard procedure to determine total expression of CITED41,2,8. However, in this study, we were able to demonstrate the difference in the spatial distribution of CITED4 expression in the six different AHA regions of the heart and in the three different transmural layers using RNA-FISH. Specifically, increases in CITED4 in the lateral wall correlate with cardiac remodeling revealed by DT-MRI-based microstructural changes. Although qPCR has previously shown significant increases in CITED4 expression in exercised heart tissue2.7, the use of RNA-FISH-based spatial information in this study directly linked microstructural tissue changes to local transcriptional changes. The implications of this CITED4 local expression can be used to guide more targeted dissemination of CITED4seven or other heart tissue modifiers, such as irisin33.34.

While CITED4 is known to play a role in tissue proliferation and hypertrophy2,7,8,9it is inversely regulated by the transcription factor C/EBPβ2 and manipulation of CITED4 levels has been shown to impact cardiomyocyte proliferation8. After exercise, a reduction in C/EBPβ results in increased growth and cell division2 and at the same time C/EBPβ may also be involved in the induction of the PGC1α pathway, which prevents cardiac dysfunction following hypertrophy2.8. Inhibition of CITED4 by C/EBPβ led to the hypothesis that the impact of C/EBPβ on the reduction of physiological growth could be directly related to the inhibition of CITED48. The data presented in this study, as well as recent studies, seem to further confirm this hypothesis.2.7. Additionally, this CITED4 pathway provides a potential link to microstructural changes revealed by DT-MRI. The specific helicity changes present between the exercise and sedentary groups could be explained, in part, by these exercise-induced molecular changes.

The specific role of CITED4 in regulating gene transcription appears to impact exercise-induced cardiac growth, as it has been shown to be a physiological marker of cardiac growth, which is also reflected in the increase heart wall thickness and LV mass in this study.2.8. Although further studies are needed to further elucidate this pathway, it appears that C/EBPβ and CITED4 are intrinsically linked to the production of exercise-induced cardiac growth.2,8,35. Exercise specifically upregulates CITED4, while downregulating C/EBPβ to produce beneficial cardiomyocyte growth and tissue remodeling from micro to macro and structural to functional2,8,35. In this study, DT-MRI analyzes confirm that trends of increased CITED4 expression occur in conjunction with generalized structural changes marked by increased helicity. Our results specifically indicate an increase in CITED4 expression in the left ventricular lateral wall. This upregulation of CITED4 in the lateral wall of the left ventricle could also help elucidate the mechanism of the lateral wall-specific increase in tissue volume found with DT-MRI.

Using CITED4 KO mice, we demonstrated the necessary role that CITED4 plays in cardiac microstructural changes in following exercise, thereby filling an important knowledge gap between the molecular mechanism of exercise and how it manifests. in tissue changes. In mice with a specific cardiac deletion of CITED4, there were significant differences in the helicity biomarker DT-MRI, indicating that CITED4 is required for cardiac structural changes to take place. Moreover, without active CITED4 expression, exercise resulted in no differences in these DT-MRI markers, providing evidence that CITED4 expression is required for exercise-induced structural changes. Meanwhile, we have demonstrated that active expression of CITED4 coupled with exercise, results in significant structural remodeling of cardiac tissue in both wild-type mice from cohort 1 and fl/fl mice from cohort 2. These results highlight the necessary and required role CITED4 plays in cardiac remodeling after exercise. Our results indicate that CITED4 also plays a critical role in basal helicity formation, as shown by the significant reduction in helicity in C4KO sedentary and exercise groups compared to fl/fl sedentary. Presumably, helicity should have been brought down to the same level as sedentary fl/fl, but was detrimentally reduced when cardiomyocyte-specific CITED4 was knocked out.

Finally, we want to emphasize that the new imaging technologies used in the study are generalizable and can be used to spatially link other molecular mechanisms to microstructural tissue remodeling. New imaging technologies could also be used to study other cardiovascular diseases as well as the impact of targeted molecular therapies such as mRNA. We believe that our study marks a potential new platform to examine how molecular mechanisms manifest in downstream tertiary tissue structures, which may play a critical role in how to increase the impact of molecular therapeutics.

However, this study has several potential limitations. The small sample size of the exercise and sedentary mouse groups is a possible limitation. Despite this limitation, a significant correlation was found between CITED4 upregulation and changes in myocardial microstructure. The comparison of Cohorts 1 and 2 had limitations, because in Cohort 2 we only had access to mid-ventricular slices of heart tissue. This prevented us from doing some of the same analyzes that we did for the first cohort of animals. Moreover, CITED4 knockdown in Cohort 2 was specific only to cardiomyocytes. However, this should not have impacted the results because CITED4 is expressed at very low levels in fibroblasts and other non-cardiomyocytes.7.8. Finally, the co-registration of CITED4 expression analyzed by RNAscope and helicity measured with DT-MRI could be a possible limitation. Future studies should pursue a more advanced method of co-registration between mRNA expression and DT-MRI markers.

Overall, this study confirmed the hypothesis that exercise leads to left ventricular microstructural changes in heart helicity and that CITED4 plays a necessary role in this process. We demonstrated that the expression of CITED4 after exercise differs between cardiac regions, which may explain the adaptive patterns of cardiac remodeling determined by cardiac DT-MRI. Moreover, we have shown that suppression of CITED4 expression in transgenic mice leads to complete prohibition of exercise-induced remodeling of heart microstructure. These findings serve as a fundamental basis for understanding exercise-induced hypertrophic changes at the molecular and microstructural level and motivate further evaluation of exercise and cardiovascular health. Future studies should explore the impacts of exercise on CITED4 using large animal models to better understand how exercise can influence cardiac remodeling in humans.

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