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.
Marks PC, Preda M, Henderson T, Liaw L, Lindner V, Friesel RE, Pinz IM. 2013. Interactive 3D analysis of blood vessel trees and collateral vessel volumes in magnetic resonance angiograms in the mouse ischemic hindlimb model. Open Med Imaging 7: 19-27.
Malaeb SN, Davis JM, Pinz IM, Newman JL, Dammann O, Rios M. 2014. Effect of sustained postnatal systemic inflammation on hippocampal volume and function in mice. Pediatr Res. 76: 363-369.
Knowles CJ, Cebova M, Pinz IM. 2013. Palmitate diet-induced loss of cardiac caveolin-3: a novel mechanism for lipid-induced contractile dysfunction. PLoS One 9: e61369.
Young K, Conley B, Romero D, Tweedie E, O’Neill C, Pinz I, Brogan L, Lindner V, Liaw L, Vary CP. 2012. BMP9 regulates endoglin-dependent chemokine responses in endothelial cells. Blood 120: 4263-4273.
Knowles CJ, Dionne M, Cebova M, Pinz I. 2011. Palmitate-induced translocation of caveolin-3 and endothelial nitric oxide synthase in cardiomyocytes. Online J Biol Sci. 11: 27-36.
Pinz I, Zhu M, Mende U, Ingwall JS. 2011. An Improved Isolation Procedure for Adult Mouse Cardiomyocytes. Cell Biochem Biophys. 61: 93-101.
Pinz I, Tian R, Belke D, Swanson E, Dillmann W, Ingwall JS. 2011. Compromised Myocardial Energetics in Hypertrophied Mouse Hearts Diminish the Beneficial Effect of Overexpressing SERCA2a. J Biol Chem. 286: 10163-10168.
Yang X, Kilgallen S, Andreeva V, Spicer DB, Pinz I, Friesel R. 2010. Conditional expression of Spry1 in neural crest causes craniofacial and cardiac defects. BMC Dev Biol. 10: 48.
Devlin MJ, Cloutier AM, Thomas NA, Panus DA, Lotinun S, Pinz I, Baron R, Rosen CJ, Bouxsein ML. 2010. Caloric restriction leads to high marrow adiposity and low bone mass in growing mice. J Bone Miner Res. 25: 2078-2088.
Urs S, Roudabush A, O’Neill CF, Pinz I, Prudovsky I, Kacer D, Tang Y, Liaw L, Small D. 2008. Soluble forms of the Notch ligands Delta1 and Jagged1 promote in vivo tumorigenicity in NIH3T3 fibroblasts with distinct phenotypes. Am J Pathol. 173: 865-878.