The research focus of my laboratory is on mechanisms leading to lipotoxic cardiomyopathy. With the growing incidence of obesity, and the burden this puts on the health care system, it is important to understand the mechanisms by which certain fatty acids and their metabolites change signaling pathways in cardiomyocytes that cause a decline in contractile performance. In particular, we focus on highly organized membrane micro-domains called caveolae. Caveolae are small flask-like membrane invaginations that on the intracellular membrane leaflet are lined by caveolin proteins. In the heart caveolin-1 and -3 are expressed and are responsible for maintaining caveolae structure. Using different high fat diets we investigate how a change in the membrane lipid composition affects caveolin proteins and what consequences this has for cardiac contractile performance. We utilize in vivo imaging techniques such as echocardiography and magnetic resonance imaging to measure cardiac contractile performance in the live mouse. To determine ex vivo contractile performance we use the Langendorff mode. We have determined that the loss of cardiac caveolin-3 by high fat feeding is part of the mechanism for lipid-induced cardiac contractile dysfunction. The figure below shows the intracellular localization of the caveolin proteins in isolated adult mouse cardiomyocytes from mice fed different high fat diets; MCT control diet, which is a high fat control diet containing only triglycerides, and palmitate diet containing about 11% of palmitate.
Figure 1: Palmitate-induced loss of T-tubular caveolin-3 and decreased protein levels. Intracellular localization of caveolin-1 and -3 in isolated cardiomyocytes from mice fed standard lab chow, MCT control or palmitate diet for 12 weeks. Caveolin-1 and -3 co-localize to the plasma membrane and the T-tubule system in standard diet fed mice. In MCT control diet fed mice caveolin-1 does not localize to the plasma membrane or the T-tubule system and in ~50% of the analyzed cardiomyocytes caveolin-1 signal was detected in the nucleus. MCT control diet does not change the localization or the amount of caveolin-3. In palmitate diet fed mice caveolin-1 and -3 co-localize to smaller areas of the plasma membrane, but not to the T-tubule system. Caveolin-1 also localizes to the nucleus in 50% of the cardiomyocytes imaged. Caveolin-3 is essentially absent from the T-tubule system in palmitate diet fed mice. This figure demonstrates the lipid dependence of caveolin protein localization in cardiomyocytes.
Signaling and Caveolin
A separate part of our work focuses on signaling proteins and receptors that bind to the caveolin scaffolding domain (CSD domain) in caveolin-3. This includes the insulin receptor and endothelial nitric oxides synthase (eNOS). For both proteins we can demonstrate that the activity and localization depends on the presence of caveolin-3 at the plasma membrane. This work has implications for vascular disease and for diabetes, two of the most common co-morbidities of obesity.
Figure 2: Palmitate induces translocation of cellular eNOS in HL-1 cardiomyocytes concomitant with the loss of caveolin-3. Cells treated with control conditions show localization of eNOS around the cell periphery (first row), while treatment with palmitate (0.4 mM) causes movement to the cell’s interior (second row). In addition, cells were treated with an inhibitor of the de novo ceramide synthesis pathway, myriocin (5 µM), which can prevent eNOS translocation during palmitate exposure (two bottom rows). Green = eNOS, Red = lipid, Blue = DNA, Yellow = areas of eNOS/lipid colocalization.
In our future work, we want to investigate how the lipid-induced loss of caveolin proteins can be prevented and what a potential pharmacological treatment to replace caveolin-3 in the heart may entail.
Small Animal Imaging Core Facility at MMCRI
The objective of the core is to provide in vivo and ex vivo imaging support to researchers throughout the New England area. The Small Animal Imaging Core Facility at MMCRI consists of a small animal magnetic resonance imager (MRI, PharmaScan, Bruker), a micro computed tomograph (µCT, Viva40, Scanco), and a high-resolution ultrasound (Vevo2100 VisualSonics).
Magnetic resonance imaging (MRI) is a non-invasive method that is utilized to look at anatomy and physiology in mice. Our small animal MR imager has a field strength of 7T and a resonance frequency of 300 MHz imager with a resolution of less than 100 µm, and is strictly proton imaging. Complimentary to the in vivo MR imaging capacity, we offer high resolution imaging with our µCT system (11 µm resolution). The µCT is located inside our barrier facility and is accessible to external users for ex vivo specimen imaging.
The high-resolution ultrasound is used for various in vivo measurements ranging from mouse pup detection to blood flow measurements, and echocardiography. This service is available to in house users and to investigators willing to order vendor approved mice for their study.
The Core operates on a fee-for-service basis and fees are established which will allow cost efficiency to investigators.
- Anatomical/morphological images of all organs including lung tumors.
- Magnetic resonance angiograms of larger blood vessels. Examples include the mouse hindlimb, lower abdomen, coronary arteries of the heart.
Figure 3: Magnetic resonance angiogram of the ischemic mouse hindlimb and corresponding µCT image of the microfill vessel cast. A) Magnetic resonance angiogram of the mouse hindlimb 7 days post right femoral artery and vein occlusion and excision. The green arrows point to the proximal and distal occlusion site. B) 3D reconstruction of the same angiogram allowing for better visualization of the collateral vessels bridging the ischemic site (red arrowheads). C) Corresponding µCT image of the same mouse showing the collateral formation in higher resolution (11 µm).
- Total body fat measurements using proton spectroscopy.
- Marrow fat measurements in long bones in vivo and ex vivo using localized spectroscopy
Figure 4: Bone marrow fat determination in the femur. The color-coding in the figure highlights high densities of protons(red areas), e.g. fat in bone marrow and water in the bladder. Localized 1H-spectrocopy is used to quantify the fat:water ratios.
- Cardiac images, diastolic and systolic dimensions of the ventricle, cardiac movies.
- In-utero assessment of embryo development/embryonic defects or determination of the time point for the loss/resorption of transgenic embryos.
- High resolution brain imaging, in vivo and ex vivo.
Figure 5: Long axis four chamber view of the mouse heart in vivo using the bright blood method. The left coronary artery can be seen as white line in the muscle where the line of the left ventricle label ends; High-resolution mouse brain image. The brain was fixed with PFA and and was imaged at a resolution of 38 µm. The image was optimized for the hippocampus area. Collaborative effort with S. Malaeb, Tufts University, Assistant Professor of Pediatrics, Division of Newborn Medicine, The Floating Hospital for Children at Tufts Medical Center.
A Service Request Form can be downloaded from the MMCRI website. Please use the link below. The form must be filled out completely with as much detailed information as possible. The completed form must include the signature and account number of the Principle Investigator of the lab requesting services. Completed forms should be submitted to Ilka Pinz.
Service Request Form