Cardiac Physiology

Cardiovascular disease is the leading cause of morbidity and mortality in the US, and sex differences are well established. Cardiovascular disease is often characterized by inappropriate cardiomyocyte death, and because adult cardiomyocytes are post-mitotic cells which undergo very limited cell division, cardiomyocyte death as occurs during myocardial infarction has very detrimental consequences. In spite of considerable effort, the mechanisms responsible for cardiomyocyte death are poorly understood. This lack of mechanistic understanding is a major contributor to the failure to translate cardioprotective drugs to the clinic. The overall goal of the research effort of my laboratory is to use a multipronged, interactive approach to elucidate the mechanisms responsible for cardiomyocyte death and to define cardioprotective strategies.

It is widely hypothesized that an increase in cytosolic calcium initiates cell death by entering mitochondria on the mitochondrial calcium uniporter (MCU) to activate a large conductance channel in the inner mitochondrial membrane known as the permeability transition pore (PTP) and that opening of this pore leads to necroptosis, a regulated form of necrotic cell death (Bauer and Murphy Circ Res 2020). Strategies to reduce PTP opening either by inhibiting the PTP directly or inhibiting the rise in its activator, mitochondrial calcium, have been proposed. A major limitation of developing strategies to inhibit the PTP is the lack of knowledge about the identity of the protein(s) that form the PTP.  However, it is agreed that  cyclophilin D (CypD), a matrix mitochondrial protein is an activator of PTP. To gain insight into the PTP we are studying the role of CypD in activating PTP.  We have defined several novel post-translational modifications (PTM) of CypD and demonstrate that these PTMs integrate multiple signaling pathways which regulate the activity of the PTP (Amanakis Cardiovas. Res 2020). We also examined the role of isomerase activity of CypD in regulating the activity of the PTP (Casin JMCC 2023).

A primary focus of the lab centers on the regulation of mitochondrial calcium, a key activator of the PTP and cell death. We published several studies examining the role of MCU and its regulatory proteins, MICU1 (Liu et al Cell Rep 2016), MICU3 (Puente Circ Res 2020) and EMRE (Liu et al JCI Insight 2020) in controlling mitochondrial calcium and ischemia-reperfusion (I/R) injury. Because an increase in mitochondrial calcium uptake via the MCU during ischemia has been proposed to active the mitochondrial PTP,  it has been hypothesized that inhibition of MCU is a viable cardioprotective approach. However there are few measurements of mitochondrial calcium during ischemia and none in an intact beating heart. To rigorously test this hypothesis we developed a new optical spectroscopy method to transmurally measure mitochondrial calcium in a beating perfused heart using mitochondrial targeted fluorescent calcium indicators (Kosmach Cell Report 2021). This new methods allow us to directly test the role of MCU and its regulators in regulating matrix calcium and the role of matrix calcium in activating the PTP. Matrix calcium levels are measured with the genetically encoded, red fluorescent calcium indicator (R-GECO1) using an adeno-associated viral vector (AAV9) for delivery. Our results demonstrate that deletion of MCU significantly reduces, but does not eliminate the accumulation of mitochondrial calcium during I/R. Elevations in mitochondrial calcium occurred in both MCU-WT and KO hearts throughout ischemia, which suggests the contribution of MCU-independent mechanisms to mitochondrial calcium overload during I/R (Petersen et al Cell Report 2023). Furthermore, our data show that increases in mitochondrial calcium occurs during ischemia. Thus, therapeutics to block increases in mitochondrial calcium and reduce PTP mediated cell death, would need to be given prior to ischemic onset to be most effective. Interestingly in WT hearts we found significantly less increase in mitochondrial calcium during ischemia in females compared to males. The mechanism responsible for this sex difference is under investigation.  We are also pursuing studies to investigate the MCU-independent mechanisms responsible for the increase in mitochondrial calcium during ischemia. These studies integrate cutting edge technologies to push back the frontiers of our understanding of mechanisms of cardiomyocyte death in cardiovascular disease.