Su Wang

Glycogen synthase kinase-3β mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3β (GSK-3β). Alternatively, receptor tyrosine kinase or certain G protein–coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3β, via protein kinase B/Akt and mTOR/p70s6k pathways, PKC pathways, or protein kinase A pathways. The convergence of these pathways via inhibition of GSK-3β on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection.

PKC-α regulates cardiac contractility and propensity toward heart failure

The protein kinase C (PKC) family of serine/threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways. Here we identify PKC-α as a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes. Hearts of Prkca-deficient mice are hypercontractile, whereas those of transgenic mice overexpressing Prkca are hypocontractile. Adenoviral gene transfer of dominant-negative or wild-type PKC-α into cardiac myocytes enhances or reduces contractility, respectively. Mechanistically, modulation of PKC-α activity affects dephosphorylation of the sarcoplasmic reticulum Ca2+ ATPase-2 (SERCA-2) pump inhibitory protein phospholamban (PLB), and alters sarcoplasmic reticulum Ca2+ loading and the Ca2+ transient. PKC-α directly phosphorylates protein phosphatase inhibitor-1 (I-1), altering the activity of protein phosphatase-1 (PP-1), which may account for the effects of PKC-α on PLB phosphorylation. Hypercontractility caused by Prkca deletion protects against heart failure induced by pressure overload, and against dilated cardiomyopathy induced by deleting the gene encoding muscle LIM protein (Csrp3). Deletion of Prkca also rescues cardiomyopathy associated with overexpression of PP-1. Thus, PKC-α functions as a nodal integrator of cardiac contractility by sensing intracellular Ca2+ and signal transduction events, which can profoundly affect propensity toward heart failure.

Role of Glycogen Synthase Kinase-3β in Cardioprotection

Limitation of infarct size by ischemic/pharmacological pre- and postconditioning involves activation of a complex set of cell-signaling pathways. Multiple lines of evidence implicate the mitochondrial permeability transition pore (mPTP) as a key end effector of ischemic/pharmacological pre- and postconditioning. Increasing the ROS threshold for mPTP induction enhances the resistance of cardiomyocytes to oxidant stress and results in infarct size reduction. Here, we survey and synthesize the present knowledge about the role of glycogen synthase kinase (GSK)-3β in cardioprotection, including pre- and postconditioning. Activation of a wide spectrum of cardioprotective signaling pathways is associated with phosphorylation and inhibition of a discrete pool of GSK-3β relevant to mitochondrial signaling. Therefore, GSK-3β has emerged as the integration point of many of these pathways and plays a central role in transferring protective signals downstream to target(s) that act at or in proximity to the mPTP. Bcl-2 family proteins and mPTP-regulatory elements, such as adenine nucleotide translocator and cyclophilin D (possibly voltage-dependent anion channel), may be the functional downstream target(s) of GSK-3β. Gaining a better understanding of these interactions to control and prevent mPTP induction when appropriate will enable us to decrease the negative impact of the reperfusion-induced ROS burst on the fate of mitochondria and perhaps allow us to limit propagation of damage throughout and between cells and consequently, to better limit infarct size.

The identity and regulation of the mitochondrial permeability transition pore: Where the known meets the unknown

The mitochondrial permeability transition (MPT) pore complex is a key participant in the machinery that controls mitochondrial fate and, consequently, cell fate. The quest for the pore identity has been ongoing for several decades and yet the main structure remains unknown. Established “dogma” proposes that the core of the MPT pore is composed of an association of voltage‐dependent anion channel (VDAC) and adenine nucleotide translocase (ANT). Recent genetic knockout experiments contradict this commonly accepted interpretation and provide a basis for substantial revision of the MPT pore identity. There is now sufficient evidence to exclude VDAC and ANT as the main pore structural components. Regarding MPT pore regulation, the role of cyclophilin D is confirmed and ANT may still serve some regulatory function, although the involvement of hexokinase II and creatine kinase remains unresolved. When cell protection signaling pathways are activated, we have found that the Bcl‐2 family members relay the signal from glycogen synthase kinase‐3 beta onto a target at or in close proximity to the pore. Our experimental findings in intact cardiac myocytes and neurons indicate that the current “dogma” related to the role of Ca2+ in MPT induction requires reevaluation. Emerging evidence suggests that after injury‐producing stresses, reactive oxygen species (but not Ca2+) are largely responsible for the pore induction. In this article we discuss the current state of knowledge and provide new data related to the MPT pore structure and regulation.

The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development

In adult myocardium, the heartbeat originates from the sequential activation of ionic currents in pacemaker cells of the sinoatrial node. Ca2+ release via the ryanodine receptor (RyR) modulates the rate at which these cells beat. In contrast, the mechanisms that regulate heart rate during early cardiac development are poorly understood. Embryonic stem (ES) cells can differentiate into spontaneously contracting myocytes whose beating rate increases with differentiation time. These cells thus offer an opportunity to determine the mechanisms that regulate heart rate during development. Here we show that the increase in heart rate with differentiation is markedly depressed in ES cell-derived cardiomyocytes with a functional knockout (KO) of the cardiac ryanodine receptor (RyR2). KO myocytes show a slowing of the rate of spontaneous diastolic depolarization and an absence of calcium sparks. The depressed rate of pacemaker potential can be mimicked in wild-type myocytes by ryanodine, and rescued in KO myocytes with herpes simplex virus (HSV)-1 amplicons containing full-length RyR2. We conclude that a functional RyR2 is crucial to the progressive increase in heart rate during differentiation of ES cell-derived cardiomyocytes, consistent with a mechanism that couples Ca2+ release via RyR before an action potential with activation of an inward current that accelerates membrane depolarization.

Matching ATP Supply and Demand in Mammalian Heart: In Vivo, In Vitro and In Silico Perspectives

Although the heart rapidly adapts cardiac output to match the bodys circulatory demands, the regulatory mechanisms ensuring that sufficient ATP is available to perform the required cardiac work are not completely understood. Two mechanisms have been suggested to serve as key regulators: (1) ADP and Pi concentrations—ATP utilization/hydrolysis in the cytosol increases ADP and Pi fluxes to mitochondria and hence the amount of available substrates for ATP production increases; and (2) Ca2+ concentration—ATP utilization/hydrolysis is coupled to changes in free cytosolic calcium and mitochondrial calcium, the latter controlling Ca2+‐dependent activation of mitochondrial enzymes taking part in ATP production. Here we discuss the evolving perspectives of each of the putative regulatory mechanisms and the precise molecular targets (dehydrogenase enzymes, ATP synthase) based on existing experimental and theoretical evidence. The data synthesis can generate novel hypotheses and experimental designs to solve the ongoing enigma of energy supply–demand matching in the heart.

Analysis of Mitochondrial 3D-Deformation in Cardiomyocytes during Active Contraction Reveals Passive Structural Anisotropy of Orthogonal Short Axes

The cardiomyocyte cytoskeleton, composed of rigid and elastic elements, maintains the isolated cell in an elongated cylindrical shape with an elliptical cross-section, even during contraction-relaxation cycles. Cardiomyocyte mitochondria are micron-sized, fluid-filled passive spheres distributed throughout the cell in a crystal-like lattice, arranged in pairs sandwiched between the sarcomere contractile machinery, both longitudinally and radially. Their shape represents the extant 3-dimensional (3D) force-balance. We developed a novel method to examine mitochondrial 3D-deformation in response to contraction and relaxation to understand how dynamic forces are balanced inside cardiomyocytes. The variation in transmitted light intensity induced by the periodic lattice of myofilaments alternating with mitochondrial rows can be analyzed by Fourier transformation along a given cardiomyocyte axis to measure mitochondrial deformation along that axis. This technique enables precise detection of changes in dimension of ∼1% in ∼1 µm (long-axis) structures with 8 ms time-resolution. During active contraction (1 Hz stimulation), mitochondria deform along the length- and width-axes of the cell with similar deformation kinetics in both sarcomere and mitochondrial structures. However, significant deformation anisotropy (without hysteresis) was observed between the orthogonal short-axes (i.e., width and depth) of mitochondria during electrical stimulation. The same degree of deformation anisotropy was also found between the myocyte orthogonal short-axes during electrical stimulation. Therefore, the deformation of the mitochondria reflects the overall deformation of the cell, and the apparent stiffness and stress/strain characteristics of the cytoskeleton differ appreciably between the two cardiomyocyte orthogonal short-axes. This method may be applied to obtaining a better understanding of the dynamic force-balance inside cardiomyocytes and of changes in the spatial stiffness characteristics of the cytoskeleton that may accompany aging or pathological conditions.

Paracrine effects of hypoxic fibroblast-derived factors on the MPT-ROS threshold and viability of adult rat cardiac myocytes.

Cardiac fibroblasts contribute to multiple aspects of myocardial function and pathophysiology. The pathogenetic relevance of cytokine production by these cells under hypoxia, however, remains unexplored. With the use of an in vitro cell culture model, this study evaluated cytokine production by hypoxic cardiac fibroblasts and examined two distinct effects of hypoxic fibroblast-conditioned medium (HFCM) on cardiac myocytes and fibroblasts. Hypoxia caused a marked increase in the production of tumor necrosis factor (TNF)-alpha by cardiac fibroblasts. HFCM significantly enhanced the susceptibility of cardiac myocytes to reactive oxygen species (ROS)-induced mitochondrial permeability transition (MPT), determined by high-precision confocal line-scan imaging following controlled, photoexcitation-induced ROS production within individual mitochondria. Furthermore, exposure of cardiac myocytes to HFCM for 5 h led to loss of viability, as evidenced by change in morphology and annexin staining. HFCM also decreased DNA synthesis in cardiac fibroblasts. Normoxic fibroblast-conditioned medium spiked with TNF-alpha at 200 pg/ml, a concentration comparable to that in HFCM, promoted loss of myocyte viability and decreased DNA synthesis in cardiac fibroblasts. These effects of HFCM are similar to the reported effects of hypoxia per se on these cell types, showing that hypoxic fibroblast-derived factors may amplify the distinct effects of hypoxia on cardiac cells. Importantly, because both hypoxia and oxidant stress prevail in a setting of ischemia and reperfusion, the effects of soluble factors from hypoxic fibroblasts on the MPT-ROS threshold and viability of myocytes may represent a novel paracrine mechanism that could exacerbate ischemia-reperfusion injury to cardiomyocytes.

Between a Rock and a Hard Place: Mitochondria Deform Anisotropically in Intact Cardiomyocytes During Active Contraction

The cardiomyocyte cytoskeleton, composed of rigid and elastic elements, maintains the shape of an elongated cylinder with an eccentrically-ellipsoidal cross-section, even during contraction-relaxation cycles. Mitochondria are micron-sized fluid-filled passive spheres distributed throughout the cardiomyocyte in a crystal-like lattice in pairs sandwiched between the sarcomere contractile machinery, longitudinally and radially; thus, their shape represents the balance of forces in 3D extant at any given moment. We developed a novel method to examine the average deformation of mitochondrial dimensions in 3D, in response to cardiomyocyte contraction and relaxation, to understand how dynamic forces are balanced inside the cardiomyocytes. The optical contrast provided by the periodic lattice of myofilaments alternating with rows of mitochondria was analyzed by examining the appropriate peaks in the frequency spectrum image along the respective cardiomyocyte axes. This technique enables precise resolution of changes in dimension of ∼1% in ∼1 µm (long axis) structures with a time resolution of 8 msec.During active contraction (1 Hz stimulation) the mitochondria deform along the length-and width-axes with similar time-to-peak deformation and 50% and 90% deformation duration characteristics in both sarcomere and mitochondrial structures. However, significant deformation anisotropy was observed between the orthogonal short (i.e., width & depth) axes of mitochondria during electrical stimulation. Interestingly, the same degree of deformation anisotropy was found between the myocyte orthogonal short axes during the same electrical stimulation; therefore, the mitochondria reflect the overall cell behavior, and the apparent stiffness and stress/strain characteristics of the cytoskeleton differ appreciably between the cardiomyocyte orthogonal short axes. This method may be applied to obtaining a better understanding of the dynamic force-balance inside cardiomyocytes and of changes in the cytoskeleton spatial stiffness characteristics that may accompany aging or pathological conditions.