Recent studies have documented that oxidized low-density lipoprotein cholesterol (oxLDL) levels directly impact myocardial structure and function. However, the molecular mechanisms by which oxLDL affects cardiac myocytes are not well established. We addressed the question whether oxLDL modifies load-free cell shortening, a standardized readout of cardiac cellular function, and investigated whether proprotein convertase subtilisin/kexin-9 (PCSK9) is involved on oxLDL-dependent processes. Adult rat ventricular cardiomyocytes were isolated and incubated for 24 h with oxLDL. PCSK9 was silenced by administration of siRNA. Load-free cell shortening was analyzed via a line camera at a beating frequency of 2 Hz. RT-PCR and immunoblots were used to identify molecular pathways. We observed a concentration-dependent reduction of load-free cell shortening that was independent of cell damage (apoptosis, necrosis). The effect of oxLDL was attenuated by silencing of oxLDL receptors (LOX-1), blockade of p38 MAP kinase activation, and silencing of PCSK9. oxLDL increased the expression of PCSK9 and caused oxidative modification of tropomyosin. In conclusion, we found that oxLDL significantly impaired contractile function via induction of PCSK9. This is the first report about the expression of PCSK9 in adult terminal differentiated ventricular cardiomyocytes. The data are important in the light of recent development of PCSK9 inhibitory strategies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-017-0650-1) contains supplementary material, which is available to authorized users.

Aims The role of uncoupling protein 2 (UCP2) in cardiac adaptation to pressure overload remains unclear. In a classical model of left ventricular pressure overload genetic deletion of UCP2 (UCP2 −/− ) protected against cardiac hypertrophy and failure. However, in UCP2 −/− mice increased proliferation of pulmonary arterial smooth muscle cells induces mild pulmonary hypertension, right ventricular (RV) hypertrophy, and reduced cardiac output. This suggests a different role for UCP2 in RV and left ventricular adaptation to pressure overload. To clarify this situation in more detail UCP2 −/− and wild-type mice were exposed to pulmonary arterial banding (PAB). Methods and results Mice were analysed (haemodynamics, morphometry, and echocardiography) 3 weeks after PAB or sham surgery. Myocytes and non-myocytes were isolated and analysed separately. Cell shortening of myocytes and fura-2 loading of cardiomyocytes were used to characterize their function. Brd assay was performed to study fibroblast proliferation. Isolated mitochondria were analysed to investigate the role of UCP2 for reactive oxygen species (ROS) production. UCP2 mRNA was 2.7-fold stronger expressed in RV myocytes than in left ventricular myocytes and stronger expressed in non-myocytes compared with myocytes. Three weeks after PAB, cardiac output was reduced in wild type but preserved in UCP2 −/− mice. UCP2 −/− had increased RV wall thickness, but lower RV internal diameters and displayed a significant stronger fibrosis. Cardiac fibroblasts from UCP2 −/− had reduced proliferation rates but higher collagen-1 expression. Myocytes isolated from mice after PAB banding showed preserved function that was further improved by UCP2 −/− . Mitochondrial ROS production and respiration was similar between UCP2 −/− or wild-type hearts. Conclusion Despite a mild pulmonary hypertension in UCP2 −/− mice, hearts from these mice are well preserved against additional pressure overload (severe pulmonary hypertension). This—at least in part—depends on different behaviour of non-myocytes (fibroblasts).

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.

Neuronal apoptosis regulated convertase-1 (NARC-1), now mostly known as proprotein convertase subtilisin/kexin type 9 (PCSK9), has received a lot of attention due to the fact that it is a key regulator of the low-density lipoprotein (LDL) receptor (LDL-R) and is therefore involved in hepatic LDL clearance. Within a few years, therapies targeting PCSK9 have reached clinical practice and they offer an additional tool to reduce blood cholesterol concentrations. However, PCSK9 is almost ubiquitously expressed in the body but has less well-understood functions and target proteins in extra hepatic tissues. As such, PCSK9 is involved in the regulation of neuronal survival and protein degradation, it affects the expression of the epithelial sodium channel (ENaC) in the kidney, it interacts with white blood cells and with cells of the vascular wall, and it modifies contractile activity of cardiomyocytes, and contributes to the regulation of cholesterol uptake in the intestine. Moreover, under stress conditions, signals from the kidney and heart can affect hepatic expression and thereby the plasma concentration of PCSK9 which then in turn can affect other target organs. Therefore, there is an intense relationship between the local (autocrine) and systemic (endocrine) effects of PCSK9. Although, PCSK9 has been recognized as a ubiquitously expressed modifier of cellular function and signaling molecules, its physiological role in different organs is not well-understood. The current review summarizes these findings.

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.