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Voltage dependent anion channel 2 (VDAC2) is an outer mitochondrial membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. However, the specific role of VDAC2 in intracellular calcium dynamics and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac ventricular myocyte-specific developmental deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium causes severe impairment in excitation-contraction coupling by altering both intracellular and mitochondrial calcium signaling. We also observed adverse cardiac remodeling which progressed to severe cardiomyopathy and death. Reintroduction of VDAC2 in 6-week-old knock-out mice partially rescued the cardiomyopathy phenotype. Activation of VDAC2 by efsevin increased cardiac contractile force in a mouse model of pressure-overload induced heart failure. In conclusion, our findings demonstrate that VDAC2 plays a crucial role in cardiac function by influencing cellular calcium signaling. Through this unique role in cellular calcium dynamics and excitation-contraction coupling VDAC2 emerges as a plausible therapeutic target for heart failure., The authors found that VDAC2 plays a crucial role in influencing mitochondrial calcium dynamics and cellular calcium signalling. A VDAC2 agonist, efsevin, rescued the heart failure phenotype, identifying a new potential therapeutic target for heart failure.

Voltage dependent anion channel 2 (VDAC2) is a mitochondrial outer membrane porin known to play a significant role in apoptosis and calcium signaling. Abnormalities in cellular calcium homeostasis often leads to electrical and contractile dysfunction and can cause dilated cardiomyopathy and heart failure. Previous literature suggests that improving mitochondrial calcium uptake via VDAC2 rescues arrhythmia phenotypes in genetic models of impaired cellular calcium signaling. However, the direct role of VDAC2 in intracellular calcium signaling and cardiac function is not well understood. To elucidate the role of VDAC2 in calcium homeostasis, we generated a cardiac-specific deletion of Vdac2 in mice. Our results indicate that loss of VDAC2 in the myocardium during development causes severe impairment in excitation-contraction coupling by reducing mitochondrial calcium uptake (n=3, p0 ) and rate of calcium uptake by SERCA2a [tau(msec)] compared to control mice (N=3, WT=54, KO=38, p0 ) and p

Pyruvate kinase is a crucial enzyme responsible for the last step of glycolysis. Cancer cells can use the M2 isoform of pyruvate kinase (PKM2), to better balance respiration and biosynthesis due to allosteric switching between the less active dimeric and fully active tetrameric forms. Additionally, the dimeric form of PKM2 can translocate to the nucleus, altering transcription to enhance cancer cells' ability to grow and evade immune detection. Inducing tetramerization presents an opportunity to target PKM2 resulting in the metabolic reprogramming of tumor-immune microenvironment (TME). TP-1454 is a potent PKM2 activator with low nanomolar PKM2 activation in biochemical assays (AC50 = 10 nM) and multiple cell types (AC < 50 nM), tolerated in mice, rats and dogs after repeat doses as high as 1000 mg/kg/day and has recently entered a Phase I clinical trial (NCT04328740). We hypothesize that PKM2 activation may reverse the immune-suppressive TME. To test this hypothesis, we examined the activity of TP-1454 combination with immunotherapy (I/O) in multiple mouse syngeneic tumor models. TP-1454 and anti-PD-1 combination therapy in colorectal cancer models resulted in tumor growth inhibition versus vehicle (53% in CT26; 99% in MC38, P < 0.001). We observed decreases in multiple glycolytic intermediates in TP-1454-treated tumors versus vehicle. We conducted immunophenotyping of the TME in multiple models to identify targets of PKM2 activation. TP-1454 treatment reduced the CD4+ Foxp3+ T-regulatory (Treg) population in MC38, 4T1, RENCA models. Further, we assayed TP-1454 induced PKM2 activation in different immune cell types. To confirm the effect of PKM2 activation on Treg cells we conducted an in vitro assay to explore TP-1454 treatment response on polarization of Tregs and/or toxicity and proliferation. We further utilized LCMS to explore metabolic intermediates that play a critical role in Treg regulation, including regulation of the O-linked β-N-acetylglucosamine (O-GlcNac) post-translational modification, which is reported to stabilize Foxp3 in CD4+ cells. We are currently exploring the effect of TP-1454 treatment on O-GlcNac of Foxp3 and its stability in HEK293 cells, to support the link between PKM2 activation and stabilization of Foxp3. TP-1454 effects on tumor-specific immunity were validated using tumor rechallenge studies. The results of a tumor rechallenge study will be presented using murine MC38 or RENCA xenograft models that are treated with TP-1454 and I/O combination therapies that exhibited a complete response (CR) and were re-implanted. These preclinical studies indicate a unique mechanism modulating tumor metabolism and the TME to improve the response of cancer patients to immunotherapy. Citation Format: Salah Sommakia, Satya Pathi, Yuta Matsumura, Curtis Allred, Ethika Tyagi, Matthew Lalonde, Jason Foulks, Adam Siddiqui, Clifford Whatcott, David Bearss, Steven Warner. Pkm2 activation modulates the tumor-immune microenvironment and enhances response to checkpoint inhibitors in preclinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 606.

The reactive tissue response of the brain to chronically implanted materials remains a formidable obstacle to stable recording from implanted microelectrodes. One approach to mitigate this response is to apply a bioactive coating in the form of an ultra-porous silica sol-gel, which can be engineered to improve biocompatibility and to enable local drug delivery. The first step in establishing the feasibility of such a coating is to investigate the effects of the coating on electrode properties. In this paper, we describe a method to apply a thin-film silica sol-gel coating to silicon-based microelectrodes, and discuss the resultant changes in the electrode properties. Fluorescently labeled coatings were used to confirm coating adherence to the electrode. Cyclic voltammetry and impedance spectroscopy were used to evaluate electrical property changes. The silica sol-gel was found to successfully adhere to the electrodes as a thin coating. The voltammograms revealed a slight increase in charge carrying capacity of the electrodes following coating. Impedance spectrograms showed a mild increase in impedance at high frequencies but a more pronounced decrease in impedance at mid to low frequencies. These results demonstrate the feasibility of applying silica sol-gel coatings to silicon-based microelectrodes and are encouraging for the continued investigation of their use in mitigating the reactive tissue response.

The reactive response of brain tissue to implantable intracortical microelectrodes is thought to negatively affect their recordable signal quality and impedance, resulting in unreliable longitudinal performance. The relationship between the progression of the reactive tissue into a glial scar and the decline in device performance is unclear. We show that exposure to a model protein solution in vitro and acute implantation result in both resistive and capacitive changes to electrode impedance, rather than purely resistive changes. We also show that applying 4000 MW polyethylene glycol (PEG) prevents impedance increases in vitro, and reduces the percent change in impedance in vivo following implantation. Our results highlight the importance of considering the contributions of non-cellular components to the decline in neural microelectrode performance, and present a proof of concept for using a simple dip-coated PEG film to modulate changes in microelectrode impedance.

The ability to design long-lasting intracortical implants hinges on understanding the factors leading to the loss of neuronal density and the formation of the glial scar. In this study, we modify a common in vitro mixed cortical culture model using lipopolysaccharide (LPS) to examine the responses of microglia, astrocytes, and neurons to microwire segments. We also use dip-coated polyethylene glycol (PEG), which we have previously shown can modulate impedance changes to neural microelectrodes, to control the cellular responses. We find that microglia, as expected, exhibit an elevated response to LPS-coated microwire for distances of up to 150 μm, and that this elevated response can be mitigated by co-depositing PEG with LPS. Astrocytes exhibit a more complex, distance-dependent response, whereas neurons do not appear to be affected by the type or magnitude of glial response within this in vitro model. The discrepancy between our in vitro responses and typically observed in vivo responses suggest the importance of using a systems approach to understand the responses of the various brain cell types in a chronic in vivo setting, as well as the necessity of studying the roles of cell types not native to the brain. Our results further indicate that the loss of neuronal density observed in vivo is not a necessary consequence of elevated glial activation.

The successful use of implantable neural microelectrodes as neuroprosthetic devices depends on the mitigation of the reactive tissue response of the brain. One of the factors affecting the ultimate severity of the reactive tissue response and the in vivo electrical properties of the microelectrodes is the initial adsorption of proteins onto the surface of the implanted microelectrodes. In this study we quantify the increase in microelectrode impedance magnitude at physiological frequencies following electrode immersion in a 10% bovine serum albumin (BSA) solution. We also demonstrate the efficacy of a common antifouling molecule, poly(ethylene glycol) (PEG), in preventing a significant increase in microelectrode impedance. In addition, we show the feasibility of using long-duration DC voltage pulses to remove adsorbed proteins from the microelectrode surface.