Jeffery Molkentin researches the molecular mechanisms underlying heart disease, focusing on necrosis—a form of cell death—and how it is regulated by mitochondria.
My laboratory pursues several diverse projects aimed at understanding the molecular mechanisms of heart and skeletal muscle disease. Toward this end, we investigate the basic machinery that underlies cell death, particularly mitochondrial-dependent mechanisms of nonapoptotic death, such as cellular necrosis (Figure 1). Prominent diseases of both heart and skeletal muscle are affected by cellular necrosis. Identifying the genes that control this process should have a substantial impact on human health.
Molkentin Research Abstract Slideshow 1
Figure 1: Cell death pathways that normally underlie cellular apoptosis and necrosis. Apoptosis consists of an extrinsic pathway mediated by death receptors such as the tumor necrosis factor receptor (TNFR) and Fas ligand receptor, which induces DISC formation to initiate caspase-8 cleavage, leading to caspase-3 activation. Activated caspase-8 can also cleave Bid, which then interacts with the mitochondria to induce outer membrane permeability and cytochrome c (CytC) release. The second arm of apoptosis is the intrinsic pathway that relies on mitochondrial-dependent killing initiated by ROS (reactive oxygen species), calcium, and hypoxic stimuli that results in cytochrome c release and apoptosome formation. The apoptosome consists of caspase-9, Apaf1, and cytochrome c, which induces caspase-3 cleavage and activation. Apoptosis also results in release of Smac, which further permits caspase activation and release of DNA fragmentation facilitators that affect the nucleus. The Bcl2 family members Bax and Bak are the primary regulators of the intrinsic pathway. Necrosis can be programmed by induction of the mitochondrial pore, leading to increased permeability after stimulation by ROS, calcium, and other stimuli. Cyclophilin D (CypD) is the only known bona fide component of this inducible pore in the mitochondria that programs necrosis.
Image: Jeffery Molkentin
Figure 2: Schematic of the intracellular signaling pathways that participate in the regulation of hypertrophy and/or disease of the heart.
Figure 3: Fibroblasts transdifferentiate into myofibroblasts in response to cytokines, where they mediate tissue repair and fibrosis. We showed that the Ca2+ channel TRPC6 provides Ca2+ to activate phosphatase calcineurin, which is then necessary and sufficient for myofibroblast differentiation through a signaling pathway that uses noncanonical transforming growth factor β (TGFβ) to mobilize p38 mitogen-activated protein kinase (MAPK) and the transcription factor SRF.
(EMC = extracellular matrix)
Image by Jeffery Molkentin. See also Davis, J. et al. 2012. Developmental Cell 23:705-715.
Figure 4: Thrombospondin (Thbs) proteins are induced during tissue damage or active remodeling in coordination with the endoplasmic reticulum (ER) stress response. We recently showed a novel function for Thbs proteins as ER resident effectors of an adaptive ER stress response. Thbs proteins bind the ER luminal domain of activating transcription factor 6α (Atf6α) to promote its processing in the Golgi and subsequent nuclear shuttling and the adaptive ER stress response. This function of Thbs secondarily affects total vesicular processing and enhances the production and secretion of many proteins, including those in the extracellular matrix (ECM) to collectively provide protection and greater resistance to various disease responses associated with ER stress and unfolded protein accumulation.
Image by Jeffery Molkentin. See also Lynch, J.M. et al. 2012. Cell 149:1257-1268.
Figure 5. GFP lineage traced heart cells from cKit lineage. Immunohistochemistry from the hearts of lineage traced mice for cKit expression. Red shows all myocyte membranes and green shows Kit allele expression, both a de novo and a fusion derived myocyte were found in the heart.
Image by Molkentin Laboratory, adapted from van Berlo, J.H. et al. 2014. Nature 509:337.
We are also interested in characterizing the intracellular signaling pathways that control cellular growth, differentiation, and replication in cardiac and skeletal muscle. We are dissecting key intracellular signaling pathways, such as calcineurin-NFAT (calcineurin is a calcium-activated phosphatase that signals to nuclear factor of activated T cells, a transcription factor) and MAPK (mitogen-activated protein kinase), as mediators of normal growth and development and disease responsiveness (Figure 2). Similarly, we are investigating the effect of novel paracrine and autocrine secreted factors that program both adaptive and maladaptive signaling effects in striated muscle. A better understanding of signaling effectors that control such processes, coupled with an identification of novel genes, could suggest new treatment strategies for human diseases.
My laboratory is also engaged in understanding the molecular identity of the mesenchymal fibroblast, especially in heart and skeletal muscle, but also in other organs and tissues that are susceptible to fibrotic diseases or wound-healing defects. We have generated a series of genetic tools in the mouse to allow lineage tracing and functional characterization of the parenchymal fibroblast to better understand the function of these cells and how we might control them in disease states or during tissue injury. Fibroblasts undergo phenotypic conversion to a related cell-type known as the myofibroblast (Figure 3), which secretes extracellular matrix and has contractile activity that mediates remodeling of injured areas of tissue. We have identified several novel effectors that regulate how myofibroblasts are formed, along with possible antifibrotic strategies that inhibit these signaling mediators.
We are also interested in understanding how unfolded proteins and the endoplasmic reticulum (ER) stress response affects disease in both heart and skeletal muscle, as well as more broadly in all tissue and cell types. We identified a novel function for the thrombospondin family of presumed extracellular matrix proteins, which in our hands show a prominent function and localization in the ER, where they directly control aspects of the ER stress response to impart cellular protection. Specifically, we showed that upregulation of thrombospondin 4 in heart or skeletal muscle leads to induction of activator transcription factor 6α to produce a protective ER stress response with expansion of the ER compartment, enhanced secretion of extracellular matrix proteins, and greater cycling and replacement of membrane proteins, as well as generally more intracellular vesicles (Figure 4). Induction of thrombospondin 4, which occurs in most injured diseased tissues, has a protective effect by initiating an adaptive ER stress response that reduces unfolded protein accumulation and helps with healing and extracellular matrix integrity.
This research has been supported in part by the National Institutes of Health.
As of February 19, 2016