Adenosine 5′ triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease.

Recent studies increasingly highlight profound metabolic remodeling within the HFpEF heart; however, it remains unclear if this is an intrinsic or systemic phenomenon. In the present study, we identify dicarbonyl and glycative stress as key drivers of mitochondrial dysfunction in an ANGII/PE mouse model with HFpEF phenotype independent of systemic metabolic disease. These findings reveal a previously unrecognized metabolic-mitochondrial axis and suggest dicarbonyl detoxification and mitochondrial antiglycation as potential therapeutic targets.

This study identifies downregulation of ACE2 in the setting of high-fat diet-induced obesity as a contributing factor to maternal cardiometabolic dysfunction and adverse pregnancy outcomes. Restoring ACE2/Ang-(1–7) function may have therapeutic value in improving cardiovascular and reproductive functions in females with obesity.

Doxorubicin (DOX) remains an effective chemotherapeutic agent, but its clinical use is limited by dose-dependent cardiotoxicity. Mitochondrial dysfunction and metabolic remodeling are central features of DOX-induced cardiac injury. ATP synthase inhibitory factor-1 (IF1) is an endogenous inhibitor of the hydrolytic activity of mitochondrial ATP synthase and has emerged as an important regulator of cellular bioenergetics. Cardiac IF1 expression is increased in multiple pathological conditions; however, its role in chemotherapy-induced cardiotoxicity remains unclear. Here, we investigated the contribution of IF1 to DOX-induced cardiotoxicity using male C57BL/6J wild-type (WT) and IF1 knockout (IF1KO) mice, isolated cardiac mitochondria, cultured neonatal cardiomyocytes, and AC16 human cardiomyocytes. Cardiac function was assessed by echocardiography, mitochondrial function by high-resolution respirometry and Seahorse metabolic flux analysis, and myocardial injury by histological and ultrastructural analyses. DOX treatment markedly increased cardiac IF1 protein levels despite reduced IF1 mRNA expression. IF1 deficiency enhanced mitochondrial respiration in isolated cardiac mitochondria and cultured cardiomyocytes under both basal and DOX-stressed conditions. IF1KO mice exhibited attenuated cardiac dysfunction and improved myocardial ultrastructure following DOX treatment compared with WT mice. In AC16 cardiomyocytes exposed to DOX, overexpression of WT IF1 improved cellular metabolic activity but provided only limited preservation of mitochondrial respiratory capacity. In contrast, overexpression of the dominant-negative IF1 mutant (IF1E30A) not only improved metabolic activity but also preserved mitochondrial respiration. These findings identify IF1 as a key regulator of metabolic adaptation during DOX stress. Upregulation of functional IF1 may represent an adaptive response that promotes glycolytic ATP production during mitochondrial stress, whereas inhibition of IF1 activity preserves metabolic activity primarily through maintenance of mitochondrial function. Collectively, these findings provide new insights into the role of IF1 in DOX-induced cardiomyopathy and highlight IF1 as a potential therapeutic target in cardio-oncology.