Sung, Jae Hwi2023-11-302023-11-302023-08https://hdl.handle.net/11299/258891University of Minnesota Ph.D. dissertation. August 2023. Major: Integrative Biology and Physiology. Advisor: Julia Liu. 1 computer file (PDF); v, 65 pages.Mitochondria are essential organelles in eukaryotic cells because they play critical roles in metabolism, intracellular ROS signaling, and cell fate decision-making. In energy-demanding organs including the heart, the role of mitochondria in energy metabolism is even more essential. To modulate mitochondrial function, Ca2+ acts as an important signaling molecule. Mitochondrial Ca2+ (mtCa2+) levels are regulated by various influx and efflux transporters such as the mitochondrial calcium uniporter complex (mtCU), the major mtCa2+ uptake pathway, and mitochondrial Na+/Ca2+/Li+ exchanger (NCLX), a major mtCa2+ efflux pathway. The mtCU is composed of the pore component MCU and regulatory components such as Mitochondrial Calcium Uptake 1-3 (MICU1-3) and Essential MCU Regulator (EMRE). MICU1 senses low cytosolic Ca2+ levels and inhibits mtCU activity as a gatekeeper. Many studies have shown that Micu1 deletion increases basal mtCa2+. EMRE is essential for MCU-mediated mtCa2+ uptake in metazoans via stabilization of the mtCU pore. Different stoichiometry of mtCU components and NCLX protein levels shape unique mtCa2+ kinetics among different types of tissues. In the heart, despite their differences in metabolism, mechanics, response to pathological stresses, and developmental origin, the right ventricle (RV) is often assumed to function similarly to the left ventricle (LV). Notably, though Ca2+ transport into mitochondria is well established to play a crucial role in matching cardiac energy production to demand and dysregulation of Ca2+ transport may contribute to fatal cardiac diseases including heart failure (HF), little is known regarding whether physiological and pathological mtCa2+ handling is distinct in the LV and RV. In addition, previous data from the lab reported that germline Micu1 deletion in mice led to mtCa2+ overload, a condition associated with multiple diseases including heart failure. At least in liver mitochondria, EMRE downregulation was reported to partially restore mtCa2+ homeostasis in MICU1 loss-mediated mtCa2+ overload, suggesting that EMRE downregulation can function as a time-dependent adaptation mechanism. However, in the heart, the adaptation mechanism to mtCa2+ overload has not been explored, and prior observation of more dramatic impairment in the RV than in the LV of mice with Micu1 deletion suggested ventricular differences that also have not been studied.To uncover whether the characteristics of mtCa2+ handling are different between the LV and RV in physiological and pathological conditions, firstly, I compared an array of parameters indicative of mtCa2+ dynamics and mitochondrial functions regulated by Ca2+ using isolated mitochondria from LV and RV free wall tissues in wild-type mice and healthy pigs. Here, I found that basal mtCa2+ levels were higher in RV mitochondria than in LV mitochondria. When successive boluses of Ca2+ were administered to isolated mitochondria, RV mitochondria took up fewer Ca2+ boluses, showing lower Ca2+ retention capacity. RV mitochondria displayed relatively more protein carbonylation, suggesting oxidative stress. Interestingly, ATP production rate was higher in RV mitochondria relative to LV mitochondria; however, only LV mitochondria exhibited an increase in ATP production rate in the presence of Ca2+. I also compared the protein expression of the subunits of the mtCU and the NCLX and found that levels of EMRE and NCLX were higher in the LV than the RV, potentially facilitating more dynamic Ca2+ transport in and out of LV mitochondria. Collectively, I found that mtCa2+ is calibrated to higher but more static levels in the RV, whereas in the LV basal mtCa2+ is lower to ensure dynamic range for physiological stimuli to increase mitochondrial bioenergetics, corresponding to the larger changes in workload experienced by the LV. Next, I compared pathological responses to mtCa2+ overload between the LV and RV as well as the possibility of time-dependent adaptation to mtCa2+ overload through downregulation of EMRE. To induce mtCa2+ overload in the heart, we generated tamoxifen (tmx)-inducible cardiac-specific Micu1 knockout mice (Micu1cKO). Here, I found by echocardiogram that LV function was reduced at 4 weeks post-tmx but was partially improved by 6 weeks post-tmx. However, RV function declined from 4 to 6 weeks post-tmx, without evidence of pulmonary arterial hypertension. To understand the differences in LV and RV response to mtCa2+ overload, we confirmed that loss of Micu1 at 1 week post-tmx resulted in mtCa2+ overload to a similar extent in both LV and RV mitochondria. Interestingly, at 7-9 weeks post-tmx, mtCa2+ and oxidative stress remained elevated in the RV mitochondria but returned to control levels in the LV mitochondria. Concurrently, EMRE protein level and mtCa2+ uptake rate were reduced in Micu1-deficient LV but not in the RV. Interestingly, in the LV, the activity of m-AAA proteases, which are known to degrade EMRE, was higher and levels of p-PKA, which can phosphorylate an m-AAA protease, were lower. Furthermore, using neonatal cardiomyocytes, I reproduced the LV-specific adaptation through EMRE downregulation in response to MICU1 loss-mediated mtCa2+ overload by using a H89, p-PKA inhibitor to augment m-AAA protease activity. Lastly, protein expression in human dilated cardiomyopathy (DCM) LV tissues suggest that a similar adaptation mechanism may occur in response to potential mtCa2+ overload induced by DCM. In summary, mtCa2+ overload results in a more pronounced impairment over time in the RV than the LV, due to compensatory EMRE reduction via increased m-AAA protease activity in the LV that is absent in the RV. To conclude, my dissertation research uncovered important differences between LV and RV cardiac mitochondria, showing that the LV maintains lower basal mtCa2+ levels to enable dynamic range in responsiveness to stimulation and that the RV is more susceptible to the mtCa2+ overload due to lack of a LV-specific adaptive response. Therefore, my studies provide new understanding of distinct ventricle-specific behaviors of LV and RV mitochondria in physiological and pathological conditions and establish a strong rationale to develop ventricular specific therapy targeting mitochondrial dysfunction.enCardiac mitochondriaMitochondriaMitochondrial calciumThesis or Dissertation