Uncoupling Proteins in Striated Muscle Tissue: Known Facts and Open Questions.

Significance: Uncoupling proteins (UCPs) are a family of proteins that allow proton leakage across the inner mitochondrial membrane. Although UCP1, also known as thermogenin, is well known and important for heat generation in brown adipose tissue, striated muscles express two distinct members of UCP, namely UCP2 and UCP3. Unlike UCP1, the main function of UCP2 and UCP3 does not appear to be heat production. Recent Advances: Interestingly, UCP2 is the main isoform expressed in cardiac tissues, whereas UCP3 is the dominant isoform in skeletal muscles. In the past years, researchers have started to investigate the regulation of UCP2 and UCP3 expression in striated muscles. Furthermore, concepts about the proposed functions of UCP2 and UCP3 in striated muscles are developed but are still a matter of debate. Critical Issues: Potential functions of UCP2 and UCP3 in striated muscles include a role in protection against mitochondria-dependent oxidative stress, as transporter for pyruvate, fatty acids, and protons into and out of the mitochondria, and in metabolic sensing. In this context, the different isoform expression of UCP2 and UCP3 in the skeletal and cardiac muscle may be related to different metabolic requirements of the two organs. Future Directions: The level of expression of UCP2 and UCP3 in striated muscles changes in different disease stages. This suggests that UCPs may become drug targets for therapy in the future. Antioxid. Redox Signal. 37, 324-335.

Untypical Metabolic Adaptations in Spontaneously Hypertensive Rats to Free Running Wheel Activity Includes Uncoupling Protein-3 (UCP-3) and Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Expression.

Obesity and hypertension are common risk factors for cardiovascular disease whereas an active lifestyle is considered as protective. However, the interaction between high physical activity and hypertension is less clear. Therefore, this study investigates the impact of high physical activity on the muscular and hepatic expression of glucose transporters (Glut), uncoupling proteins (UCPs), and proprotein convertase subtilisin/kexin type 9 (PCSK9) in spontaneously hypertensive rats (SHRs). Twenty-four female rats (12 normotensive rats and 12 SHRs) were divided into a sedentary control and an exercising group that had free access to running wheels at night for 10 months. Blood samples were taken and blood pressure was determined. The amount of visceral fat was semi-quantitatively analyzed and Musculus gastrocnemius, Musculus soleus, and the liver were excised. Acute effects of free running wheel activity were analyzed in 15 female SHRs that were sacrificed after 2 days of free running wheel activity. M. gastrocnemius and M. soleus differed in their mRNA expression of UCP-2, UCP-3, GLUT-4, and PCSK9. Hypertension was associated with lower levels of UCP-2 and PCSK9 mRNA in the M. gastrocnemius, but increased expression of GLUT-1 and GLUT-4 in the M. soleus. Exercise down-regulated UCP-3 in the M. soleus in both strains, in the M. gastrocnemius only in normotensives. In SHRs exercise downregulated the expression of UCP-2 in the M. soleus. Exercise increased the expression of GLUT-1 in the M. gastrocnemius in both strains, and that of GLUT-4 protein in the M. soleus, whereas it increased the muscle-specific expression of PCSK9 only in normotensive rats. Effects of exercise on the hepatic expression of cholesterol transporters were seen only in SHRs. As an acute response to exercise increased expressions of the myokine IL-6 and that of GLUT-1 were found in the muscles. This study, based on transcriptional adaptations in striated muscles and livers, shows that rats perform long-term metabolic adaptations when kept with increased physical activity. These adaptations are at least in part required to stabilize normal protein expression as protein turnover seems to be modified by exercise. However, normotensive and hypertensive rats differed in their responsiveness. Based on these results, a direct translation from normotensive to hypertensive rats is not possible. As genetic differences between normotensive humans and patients with essential hypertension are likely to be present as well, we would expect similar differences in humans that may impact recommendations for non-pharmacological interventions.

Lack of Contribution of p66shc to Pressure Overload-Induced Right Heart Hypertrophy.

The leading cause of death in pulmonary arterial hypertension (PAH) is right ventricular (RV) failure (RVF). Reactive oxygen species (ROS) have been suggested to play a role in the development of RV hypertrophy (RVH) and the transition to RVF. The hydrogen peroxide-generating protein p66shc has been associated with left ventricular (LV) hypertrophy but its role in RVH is unclear. The purpose of this study was to determine whether genetic deletion of p66shc affects the development and/or progression of RVH and RVF in the pulmonary artery banding (PAB) model of RV pressure overload. The impact of p66shc on mitochondrial ROS formation, RV cardiomyocyte function, as well as on RV morphology and function were studied three weeks after PAB or sham operation. PAB in wild type mice did not affect mitochondrial ROS production or RV cardiomyocyte function, but induced RVH and impaired cardiac function. Genetic deletion of p66shc did also not alter basal mitochondrial ROS production or RV cardiomyocyte function, but impaired RV cardiomyocyte shortening was observed following PAB. The development of RVH and RVF following PAB was not affected by p66shc deletion. Thus, our data suggest that p66shc-derived ROS are not involved in the development and progression of RVH or RVF in PAH.

Autocrine effects of PCSK9 on cardiomyocytes.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is in the focus of cardiovascular research due to its role in hepatic low density lipoprotein (LDL) clearance. However, extrahepatic expression of PCSK9 such as in cardiomyocytes and its regulation by oxidized LDL (oxLDL) put notion on extrahepatic effects of PCSK9 as well. This study was aimed to reveal the role of PCSK9 in oxLDL-dependent regulation of cardiomyocyte function. Adult rat and mouse ventricular cardiomyocytes and isolated perfused hearts were used. OxLDL was applied to increase PCSK9 expression in cardiomyocytes. Cell function was analyzed by load-free cell shortening as well as left ventricular developed pressure of isolated hearts. OxLDL decreased shortening in wild-type-derived mouse cardiomyocytes but not in those isolated from PCSK9 knockout mice. Overexpression of human PCSK9 in rat cardiomyocytes reduced shortening in the absence of oxLDL. Addition of recombinant PCSK9 mimicked these effects. In cardiomyocytes, oxLDL induced PCSK9 release into the supernatant. Inhibition of PCSK9 by Pep 2-8 or alirocumab attenuated the oxLDL-induced loss of cardiomyocyte shortening. Cardiomyocytes express surfeit locus protein 4 (SURF-4), a protein required for PCSK9 secretion in human embryonic kidney cells (HEK 293 T), and silencing of SURF-4 reduced the oxLDL effects on cardiomyocytes. In isolated perfused rat hearts PCSK9 inhibition by alirocumab improved the function. In addition, left ventricular function of isolated hearts from PCSK9 knockout mice was increased under basal conditions as well as at 10 min and 120 min of reperfusion following 45 min of ischemia. Collectively, the data show that cardiomyocytes express and release PCSK9 that acts in an autocrine way on cardiomyocytes and impairs their function.

Induction of Proteasome Subunit Low Molecular Weight Protein (LMP)-2 Is Required to Induce Active Remodeling in Adult Rat Ventricular Cardiomyocytes.

Isolated adult rat ventricular cardiomyocytes (ARVC) adapt to the two-dimensional surface of culture dishes once they are isolated from the three-dimensional heart tissue. This process mimics aspects of cardiac adaptation to pressure overload and requires an initial breakdown of sarcomeric structures. The present study therefore aimed to identify key steps in this remodeling process. ARVC were cultured under serum-free or serum-supplemented conditions and their sizes and shapes were analyzed as well as apoptosis and the ability to disintegrate their sarcomeres. ARVC require serum-factors in order to adapt to cell culture conditions. More ARVC survived if they were able to breakdown their sarcomeres and mononucleated ARVC, which were smaller than binucleated ARVC, had a better chance to adapt. During the early phase of adaptation, proteasome subunit low molecular weight protein (LMP)-2 was induced. Inhibition of LMP-2 up-regulation by siRNA attenuated the process of successful adaptation. In vivo, LMP-2 was induced in the left ventricle of spontaneously hypertensive rats during the early phase of adaptation to pressure overload. In conclusion, the data suggest that breakdown of pre-existing sarcomeres is optimized by induction of LMP-2 and that it is required for cardiac remodeling processes, for example, occurring during pressure overload.

Ischaemic post-conditioning in rats: Responder and non-responder differ in transcriptome of mitochondrial proteins.

Ischaemic post-conditioning (IPoC) is a clinical applicable procedure to reduce reperfusion injury. Non-responsiveness to IPoC possibly caused by co-morbidities limits its clinical attractiveness. We analysed differences in the expression of mitochondrial proteins between IPoC responder (IPoC-R) and non-responder (IPoC-NR). Eighty rats were randomly grouped to sham, ischaemia/reperfusion (I/R), IPoC or ischaemic pre-conditioning (IPC, as positive cardioprotective intervention) in vivo. Infarct sizes were quantified by plasma troponin I levels 60 minutes after reperfusion. After 7 days, rats were sacrificed and left ventricular tissue was taken for post hoc analysis. The transcriptome was analysed by qRT-PCR and small RNA sequencing. Key findings were verified by immunoblots. I/R increased plasma troponin I levels compared to Sham. IPC reduced troponin I compared to I/R, whereas IPoC produced either excellent protection (IPoC-R) or no protection (IPoC-NR). Twenty-one miRs were up-regulated by I/R and modified by IPoC. qRT-PCR analysis revealed that IPoC-R differed from other groups by reduced expression of arginase-2 and bax, whereas the mitochondrial uncoupling protein (UCP)-2 was induced in IPC and IPoC-R. IPoC-R and IPoC-NR synergistically increased the expression of non-mitochondrial proteins like VEGF and SERCA2a independent of the infarct size. Cardiac function was more closely linked to differences in mitochondrial proteins than on regulation of calcium-handling proteins. In conclusion, healthy rats could not always be protected by IPoC. IPoC-NR displayed an incomplete responsiveness which is reflected by different changes in the mitochondrial transcriptome compared to IPoC-R. This study underlines the importance of mitochondrial proteins for successful long-term outcome.

Alterations in Glucose Metabolism During the Transition to Heart Failure: The Contribution of UCP-2.

The cardiac expression of the mitochondrial uncoupling protein (UCP)-2 is increased in patients with heart failure. However, the underlying causes as well as the possible consequences of these alterations during the transition from hypertrophy to heart failure are still unclear. To investigate the role of UCP-2 mechanistically, expression of UCP-2 was silenced by small interfering RNA in adult rat ventricular cardiomyocytes. We demonstrate that a downregulation of UCP-2 by siRNA in cardiomyocytes preserves contractile function in the presence of angiotensin II. Furthermore, silencing of UCP-2 was associated with an upregulation of glucose transporter type (Glut)-4, increased glucose uptake, and reduced intracellular lactate levels, indicating improvement of the oxidative glucose metabolism. To study this adaptation in vivo, spontaneously hypertensive rats served as a model for cardiac hypertrophy due to pressure overload. During compensatory hypertrophy, we found low UCP-2 levels with an upregulation of Glut-4, while the decompensatory state with impaired function was associated with an increase of UCP-2 and reduced Glut-4 expression. By blocking the aldosterone receptor with spironolactone, both cardiac function as well as UCP-2 and Glut-4 expression levels of the compensated phase could be preserved. Furthermore, we were able to confirm this by left ventricular (LV) biopsies of patients with end-stage heart failure. The results of this study show that UCP-2 seems to impact the cardiac glucose metabolism during the transition from hypertrophy to failure by affecting glucose uptake through Glut-4. We suggest that the failing heart could benefit from low UCP-2 levels by improving the efficiency of glucose oxidation. For this reason, UCP-2 inhibition might be a promising therapeutic strategy to prevent the development of heart failure.

Review on Chamber-Specific Differences in Right and Left Heart Reactive Oxygen Species Handling.

Reactive oxygen species (ROS) exert signaling character (redox signaling), or damaging character (oxidative stress) on cardiac tissue depending on their concentration and/or reactivity. The steady state of ROS concentration is determined by the interplay between its production (mitochondrial, cytosolic, and sarcolemmal enzymes) and ROS defense enzymes (mitochondria, cytosol). Recent studies suggest that ROS regulation is different in the left and right ventricle of the heart, specifically by a different activity of superoxide dismutase (SOD). Mitochondrial ROS defense seems to be lower in right ventricular tissue compared to left ventricular tissue. In this review we summarize the current evidence for heart chamber specific differences in ROS regulation that may play a major role in an observed inability of the right ventricle to compensate for cardiac stress such as pulmonary hypertension. Based on the current knowledge regimes to increase ROS defense in right ventricular tissue should be in the focus for the development of future therapies concerning right heart failure.