Although incidence and prevalence of prediabetes are increasing, little is known about its cardiac effects. Therefore, our aim was to investigate the effect of prediabetes on cardiac function and to characterize parameters and pathways associated with deteriorated cardiac performance. Long-Evans rats were fed with either control or high-fat chow for 21 wk and treated with a single low dose (20 mg/kg) of streptozotocin at week 4 High-fat and streptozotocin treatment induced prediabetes as characterized by slightly elevated fasting blood glucose, impaired glucose and insulin tolerance, increased visceral adipose tissue and plasma leptin levels, as well as sensory neuropathy. In prediabetic animals, a mild diastolic dysfunction was observed, the number of myocardial lipid droplets increased, and left ventricular mass and wall thickness were elevated; however, no molecular sign of fibrosis or cardiac hypertrophy was shown. In prediabetes, production of reactive oxygen species was elevated in subsarcolemmal mitochondria. Expression of mitofusin-2 was increased, while the phosphorylation of phospholamban and expression of Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3, a marker of mitophagy) decreased. However, expression of other markers of cardiac auto- and mitophagy, mitochondrial dynamics, inflammation, heat shock proteins, Ca/calmodulin-dependent protein kinase II, mammalian target of rapamycin, or apoptotic pathways were unchanged in prediabetes. This is the first comprehensive analysis of cardiac effects of prediabetes indicating that mild diastolic dysfunction and cardiac hypertrophy are multifactorial phenomena that are associated with early changes in mitophagy, cardiac lipid accumulation, and elevated oxidative stress and that prediabetes-induced oxidative stress originates from the subsarcolemmal mitochondria.

Aims: Research into right ventricular (RV) physiology and identification of pathomechanisms underlying RV failure have been neglected for many years, because function of the RV is often considered less important for overall hemodynamics and maintenance of blood circulation. In view of this, this study focuses on identifying specific adaptive mechanisms of the RV and left ventricle (LV) during a state of chronic nitric oxide (NO) deficiency, one of the main causes of cardiac failure. NO deficiency was induced in rats by L-NAME feeding over a 4 week period. The cardiac remodeling was then characterized separately for the RV/LV using quantitative real-time polymerase chain reaction, histology, and functional measurements. Results: Only the RV underwent remodeling that corresponded morphologically and functionally with the pattern of dilated cardiomyopathy. Symptoms in the LV were subtle and consisted primarily of moderate hypertrophy. A massive increase in reactive oxygen species (ROS) (+4.5 -0.8-fold, vs. control) and a higher degree of oxidized tropomyosin (+46% -4% vs. control) and peroxynitrite (+32% -2% vs. control) could be identified as the cause of both RV fibrosis and contractile dysfunction. The expression of superoxide dismutase-2 was specifically increased in the LV by 51% -3% and prevented the ROS increase and the corresponding structural and functional remodeling. Innovation: This study identified the inability of the RV to increase its antioxidant capacity as an important risk factor for developing RV failure. Conclusion: Unlike the LV, the RV did not display the necessary adaptive mechanisms to cope with increased oxidative stress during a state of chronic NO deficiency. Antioxid. Redox Signal. 23, 1220-1232.

Key points• Physical exercise is recommended as first line therapy for hypertensive patients. However, studies investigating long-term effects of high intensity exercise on the progression of hypertensive heart disease have revealed conflicting results.• We show that high intensity aerobic exercise accelerates hypertensive heart disease and improves fibrosis.• Surprisingly, high intensity aerobic exercise in the presence of an angiotensin converting enzyme inhibitor not only attenuated training induced mal-adaptation but exerts positive repair processes. These effects were independent of blood pressure effects.• The results of this study provide evidence that high physical activity in hypertensives must be considered as an important risk factor rather than a therapeutic intervention. AbstractIn the present study it was hypothesized that voluntary aerobic exercise favours a pro-fibrotic phenotype and promotes adverse remodelling in hearts from spontaneously hypertensive rats (SHRs) in an angiotensin II-dependent manner. To test this, female SHRs at the age of 1 year were started to perform free running wheel exercise. Captopril was used to inhibit the renin-angiotensin system (RAS). Normotensive rats and SHRs kept in regular cages were used as sedentary controls. Training intensity, expressed as mean running velocity, was positively correlated with the left ventricular mRNA expression of TGF-β 1 , collagen-III and biglycan but negatively correlated with the ratio of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA)2a to Na + -Ca 2+ exchanger (NCX). A pro-fibrotic phenotype was verified by Picrosirius red staining. Sixty-seven per cent of SHRs performing free running wheel exercise died either spontaneously or had to be killed during a 6 month follow-up. In the presence of captopril, aerobic exercise did not show a similar positive correlation between training intensity and the expression of fibrotic markers. Moreover, in SHRs receiving captopril and performing free running wheel exercise, a training intensity-dependent reverse remodelling of the SERCA2a-to-NCX ratio was observed. None of these rats died spontaneously or had to be killed. In captopril-treated SHRs performing exercise, expression of mRNA for decorin, a natural inhibitor of TGF-β 1 , was up-regulated. Despite these differences between SHR-training groups with and without captopril, positive training effects (lower resting heart rate and no progression of hypertension) were found in both groups. In conclusion, high aerobic exercise induces an angiotensin II-dependent adverse Abbreviations ACE, angiotensin converting enzyme; NCX, Na + -Ca 2+ exchanger; Nrf-1, nuclear respiratory factor-1; PGC-1α, peroxisome proliferator activated receptor γ coactivator 1α; RAS, renin-angiotensin system; SERCA, sarcoplasmatic reticulum Ca 2+ -ATPase; SHR, spontaneously hypertensive rat; TGF-β1, transforming growth factor-β1.

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.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells. Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are β-myosin heavy chain (β-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.