The Cardozo Lab is currently focused on a number of projects related to skeletal muscle health and function both in normal muscle and muscle paralyzed by a spinal cord injury, injured by trauma or impaired by disease. The main areas of study are described below.
The roles of Numb and Numblike in skeletal muscle
Numb is a protein that has been shown to be a major regulator of asymmetric cell division. It has additional roles in inhibiting Notch signaling by targeting Notch1 for proteasomal degradation but the role of Numb in skeletal muscle is largely unknown. In satellite cells, muscle-specific stem cells, Numb is necessary for their differentiation into muscle precursor cells. Our lab has shown that Numb is protected from degradation by the anabolic steroid nandrolone through reductions in MDM2 expression and that Numb transcription is upregulated through activation of Wnt/β-catenin signaling. We have further demonstrated in C2C12 cells that Numb binds to the slow myosin isoform promoter by tethering to MyoD at MyoD binding sites.
Building upon this evidence, our lab generated an inducible, skeletal muscle-restricted floxed Numb and Numblike mouse to further investigate mechanisms through which Numb and a similar protein, Numblike, may affect skeletal muscle. We are currently investigating the functions of Numb in excitation-contraction coupling and tissue repair. More information can be found in the following PDFs of recent conference poster presentations.
Connexin hemichannels and muscle dysfunction
Connexins are the building blocks of gap junctions which serve as pores through which salts and other small molecules move between cells to propagate action potentials and facilitate inter-cellular communications. Connexins have also been found on the surface of cells as hemichannels that are not coupled to cognate connexins on adjacent cells. These hemichannels allow flow of salts, ATP and other molecules along concentration gradient when hemichannels are ‘open’. Recent evidence demonstrates that appearance on the surface of muscle fibers of connexin hemichannels is an early event in muscle atrophy as well as several other form of muscle dysfunction. A current interest in the lab is to better understand the role of such hemichannels in the pathogenesis of mitochondrial dysfunction and muscle atrophy after paralysis with an eye to using connexin hemichannel inhibitors to mitigate adverse effects of aberent hemichannel expression or opening.
Changes in metabolism after SCI
It has been well-documented that those with SCI are more prone to systemic metabolic dysfunction than the normal population. Some of this association may be explained by the loss in skeletal muscle mass due to paralysis and reductions in physical activity. Skeletal muscle takes up a large amount of glucose to store and/or use for energy. The loss of muscle mass reduces this ability and leads to higher levels of blood glucose, markers of insulin resistance, and higher rates of prediabetes and type 2 diabetes. Those with SCI are also likely to gain large amounts of both visceral and subcutaneous fat across the duration of their injury. These elevations in fat mass may exacerbate some of the metabolic risk factors those with SCI face, including low-grade systemic inflammation and cardiovascular diseases, all of which reduce well-being and potentially independent living.
Our lab is using cell culture models to show that factors secreted from C2C12 myoblasts can affect metabolic markers in fat cells or liver cells. Further, a molecule that mimics the action of adiponectin, a beneficial hormone released from adipose cells, can reduce the impact of deleterious factors secreted from skeletal muscle. Within skeletal muscle itself, we have found acute reductions in glucose and downstream glycolytic substrates at 7 days after a complete SCI in mice and this reduction is resolved by 28 days. At these timepoints we also note changes in key enzymes associated with glycolysis and small carbon molecules. Lastly, we have recently begun studies looking into how high-fat diets can affect parameters of muscle function and systemic metabolism after SCI. More information can be found in the following PDFs of recent conference poster presentations.
Role of NOX4 in disrupting muscle function after SCI
Skeletal muscle contracts following signals from the motor neuron which cause a depolarization of the muscle fiber membrane, called the sarcolemma. The sarcolemma contains invaginations called t-tubules, which penetrate into the muscle fiber. Sarcolemmal depolarization opens a voltage-gated calcium channel called the dihydropyridine receptor, or DHPr. Opening of the DHPr opens another important protein called the ryanodine receptor 1 (RyR1), a protein in the membrane of the sarcoplasmic reticulum (SR) which releases large amounts of calcium into the cytosol to bind to actin and allow for a muscle contraction. We have shown that at 56 days after a complete SCI, there is biochemical evidence of oxidative damage to the RyR1 known to cause a ‘leaky’ SR, a situation which can lead to chronic levels of excess cytosolic calcium and poor muscle function and health. NOX4 is a protein that generates reactive oxygen species (ROS) and is upregulated both in protein and mRNA expression after SCI. ROS have wide-ranging effects on cell health. We are currently conducting studies to determine how drugs that correct RyR1 function or block the action of NOX4 impact muscle function after SCI. Such agents may provide a unique approach to protect muscle health after SCI.
ApoE4 alleles and functional outcomes post-SCI
ApoE is a lipoprotein that serves to carry lipids between cells of different types. ApoE4 is one of three genetic variants of ApoE found in humans. ApoE4 is linked to increased risk of Alzheimer’s disease and cardiovascular disease. It appears to also predict worse function after spinal cord injury. We are interested in understanding the cellular and molecular basis for this genetic association with the ultimate aim of developing drugs targeting the mechanisms responsible and thus improving the lives of those so injured.