Tobias Speerschneider

Expression of interleukin-15 in human skeletal muscle – effect of exercise and muscle fibre type composition

The cytokine interleukin‐15 (IL‐15) has been demonstrated to have anabolic effects in cell culture systems. We tested the hypothesis that IL‐15 is predominantly expressed by type 2 skeletal muscle fibres, and that resistance exercise regulates IL‐15 expression in muscle. Triceps brachii, vastus lateralis quadriceps and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers (n= 14), because these muscles are characterized by having different fibre‐type compositions. In addition, healthy, normally physically active male subjects (n= 8) not involved in any kind of resistance exercise underwent a heavy resistance exercise protocol that stimulated the vastus lateralis muscle and biopsies were obtained from this muscle pre‐exercise as well as 6, 24 and 48 h post‐exercise. IL‐15 mRNA levels were twofold higher in the triceps (type 2 fibre dominance) compared with the soleus muscle (type 1 fibre dominance), but Western blotting and immunohistochemistry revealed that muscle IL‐15 protein content did not differ between triceps brachii, quadriceps and soleus muscles. Following resistance exercise, IL‐15 mRNA levels were up‐regulated twofold at 24 h of recovery without any changes in muscle IL‐15 protein content or plasma IL‐15 at any of the investigated time points. In conclusion, IL‐15 mRNA level is enhanced in skeletal muscles dominated by type 2 fibres and resistance exercise induces increased muscular IL‐15 mRNA levels. IL‐15 mRNA levels in skeletal muscle were not paralleled by similar changes in muscular IL‐15 protein expression suggesting that muscle IL‐15 may exist in a translationally inactive pool.

Lack of evidence for AT1R/B2R heterodimerization in COS-7, HEK293, and NIH3T3 cells: how common is the AT1R/B2R heterodimer?

It has been suggested previously ( AbdAlla, S., Lother, H., and Quitterer, U. (2000) Nature 407, 94-98 ) that the angiotensin II type 1 receptor (AT1R) and the bradykinin B2 receptor (B2R) form constitutive heterodimers. Furthermore they demonstrate that AT1R signaling significantly increases in the presence of the B2R. These findings suggest that heterodimerization and potentiation of AT1R signaling is a universal phenomenon that occurs as a natural consequence of simultaneous expression of the two receptors. Hence this potential interaction is of great pharmacological and biological interest that adds an additional layer of complexity to the understanding of the cross-talk between the renin-angiotensin and kallikrein-kinin systems. Given the remarkable significance of this finding, scientists from four independent research groups have set out to reproduce and further examine the potential AT1R/B2R interaction. We have investigated functional potentiation by the B2R of AT1R signaling in three different cell lines using multiple assays including phosphoinositide hydrolysis, ERK activation, β-arrestin recruitment, and receptor selection and amplification technology, and we have examined dimerization using bioluminescence resonance energy transfer and regulated secretion/aggregation technology. However, although both the AT1Rs and B2Rs were functional in our systems and the systems were fine tuned to detect small changes in receptor function, we failed to detect any functional modulation by or physical interaction between the two receptor proteins. In contrast to the previous observations, our data collectively suggest that AT1R/B2R heterodimerization does not occur as a natural consequence of their simultaneous expression in the same cell nor does the B2R influence the AT1R signaling.

Physiology and analysis of the electrocardiographic T wave in mice.

The murine electrocardiogram (ECG) is a valuable tool in cardiac research, although the definition of the T wave has been a matter of debate for several years potentially leading to incomparable data. By this study, we seek to make a clear definition of the murine T wave. Moreover, we investigate the consequences of performing QT interval correction in anaesthetized mice.

Loss of K+ Currents in Heart Failure Is Accentuated in KChIP2 Deficient Mice

KV4 together with KV Channel‐Interacting Protein 2 (KChIP2) mediate the fast recovering transient outward potassium current (Ito,f) in the heart. KChIP2 is downregulated in human heart failure (HF), potentially underlying the loss of Ito,f. We investigated remodeling associated with HF hypothesizing that KChIP2 plays a central role in the modulation of outward K+ currents in HF.

5′-AMP Activated Protein Kinase is Involved in the Regulation of Myocardial β-Oxidative Capacity in Mice

5′-adenosine monophosphate-activated protein kinase (AMPK) is considered central in regulation of energy status and substrate utilization within cells. In heart failure the energetic state is compromised and substrate metabolism is altered. We hypothesized that this could be linked to changes in AMPK activity and we therefore investigated mitochondrial oxidative phosphorylation capacity from the oxidation of long- and medium-chain fatty acids (LCFA and MCFA) in cardiomyocytes from young and old mice expressing a dominant negative AMPKα2 (AMPKα2-KD) construct and their wildtype (WT) littermates. We found a 35–45% (P < 0.05) lower mitochondrial capacity for oxidizing MCFA in AMPKα2-KD of both age-groups, compared to WT. This coincided with marked decreases in protein expression (19/29%, P < 0.05) and activity (14/21%, P < 0.05) of 3-hydroxyacyl-CoA-dehydrogenase (HAD), in young and old AMPKα2-KD mice, respectively, compared to WT. Maximal LCFA oxidation capacity was similar in AMPKα2-KD and WT mice independently of age implying that LCFA-transport into the mitochondria was unaffected by loss of AMPK activity or progressing age. Expression of regulatory proteins of glycolysis and glycogen breakdown showed equivocal effects of age and genotype. These results illustrate that AMPK is necessary for normal mitochondrial function in the heart and that decreased AMPK activity may lead to an altered energetic state as a consequence of reduced capacity to oxidize MCFA. We did not identify any clear aging effects on mitochondrial function.

The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately connected and some of the cardioprotective effects of Losartan are abolished by blocking the bradykinin B2 receptor (B2R) signaling. In this study, we investigated the ability of six clinically available ARBs to specifically bind and activate the B2R. First, we investigated their ability to activate phosphoinositide (PI) hydrolysis in COS-7 cells transiently expressing the B2R. We found that only Losartan activated the B2R, working as a partial agonist compared to the endogenous ligand bradykinin. This effect was blocked by the B2R antagonist HOE 140. A competitive binding analysis revealed that Losartan does not significantly compete with bradykinin and does not change the binding affinity of bradykinin on the B2R. Furthermore, Losartan but not Candesartan mimicked the ability of bradykinin to increase the recovery of contractile force after metabolic stress in rat atrial tissue strips. In conclusion, Losartan is a partial agonist of the B2R through direct binding and activation, suggesting that B2R agonism could partly explain the beneficial effects of Losartan.

Preservation of Cardiac Function by Prolonged Action Potentials in Mice Deficient of KChIP2

Inherited ion channelopathies and electrical remodeling in heart disease alter the cardiac action potential with important consequences for excitation-contraction coupling. Potassium channel-interacting protein 2 (KChIP2) is reduced in heart failure and interacts under physiological conditions with both Kv4 to conduct the fast-recovering transient outward K(+) current (Ito,f) and with CaV1.2 to mediate the inward L-type Ca(2+) current (ICa,L). Anesthetized KChIP2(-/-) mice have normal cardiac contraction despite the lower ICa,L, and we hypothesized that the delayed repolarization could contribute to the preservation of contractile function. Detailed analysis of current kinetics shows that only ICa,L density is reduced, and immunoblots demonstrate unaltered CaV1.2 and CaVβ₂ protein levels. Computer modeling suggests that delayed repolarization would prolong the period of Ca(2+) entry into the cell, thereby augmenting Ca(2+)-induced Ca(2+) release. Ca(2+) transients in disaggregated KChIP2(-/-) cardiomyocytes are indeed comparable to wild-type transients, corroborating the preserved contractile function and suggesting that the compensatory mechanism lies in the Ca(2+)-induced Ca(2+) release event. We next functionally probed dyad structure, ryanodine receptor Ca(2+) sensitivity, and sarcoplasmic reticulum Ca(2+) load and found that increased temporal synchronicity of the Ca(2+) release in KChIP2(-/-) cardiomyocytes may reflect improved dyad structure aiding the compensatory mechanisms in preserving cardiac contractile force. Thus the bimodal effect of KChIP2 on Ito,f and ICa,L constitutes an important regulatory effect of KChIP2 on cardiac contractility, and we conclude that delayed repolarization and improved dyad structure function together to preserve cardiac contraction in KChIP2(-/-) mice.

Development of heart failure is independent of K+ channel-interacting protein 2 expression.

•  Previous studies have suggested that the K+ channel auxiliary subunit K+ channel‐interacting protein 2 (KChIP2) serves as a regulator of cardiac remodelling leading to heart failure and increased risk of arrhythmias. •  The results presented here show that the progression of cardiac remodelling and heart failure induced by transverse aortic constriction follows a similar time course in wild‐type and KChIP2−/− mice. •  Protein expression analysis shows that ventricular KChIP2 is significantly downregulated in heart failure in wild‐type mice. •  The electrophysiological analysis reveals enlarged J and T wave amplitudes and lower vulnerability to pacing‐induced ventricular arrhythmias in KChIP2−/− control mice compared to wild‐type control mice. Heart failure in wild‐type and KChIP2−/− mice prompted comparable prolongation of QT intervals and ventricular effective refractory periods. •  Collectively, these results demonstrate that KChIP2 does not influence the structural and functional development of heart failure. Moreover, in contrast to previously reported data, downregulation of KChIP2 expression in heart failure may reduce the risk of cardiac arrhythmia.

Sinoatrial node dysfunction induces cardiac arrhythmias in diabetic mice

BackgroundThe aim of this study was to probe cardiac complications, including heart-rate control, in a mouse model of type-2 diabetes. Heart-rate development in diabetic patients is not straight forward: In general, patients with diabetes have faster heart rates compared to non-diabetic individuals, yet diabetic patients are frequently found among patients treated for slow heart rates. Hence, we hypothesized that sinoatrial node (SAN) dysfunction could contribute to our understanding of the mechanism behind this conundrum and the consequences thereof.MethodsCardiac hemodynamic and electrophysiological characteristics were investigated in diabetic db/db and control db/+ mice.ResultsWe found improved contractile function and impaired filling dynamics of the heart in db/db mice, relative to db/+ controls. Electrophysiologically, we observed comparable heart rates in the two mouse groups, but SAN recovery time was prolonged in diabetic mice. Adrenoreceptor stimulation increased heart rate in all mice and elicited cardiac arrhythmias in db/db mice only. The arrhythmias emanated from the SAN and were characterized by large RR fluctuations. Moreover, nerve density was reduced in the SAN region.ConclusionsEnhanced systolic function and reduced diastolic function indicates early ventricular remodeling in obese and diabetic mice. They have SAN dysfunction, and adrenoreceptor stimulation triggers cardiac arrhythmia originating in the SAN. Thus, dysfunction of the intrinsic cardiac pacemaker and remodeling of the autonomic nervous system may conspire to increase cardiac mortality in diabetic patients.

Ventricular repolarization time, location of pacing stimulus and current pulse amplitude conspire to determine arrhythmogenicity in mice

In this study, we investigate the impact of altered action potential durations (APD) on ventricular repolarization time and proarrhythmia in mice with and without genetic deletion of the K+‐channel‐interacting protein 2 (KChIP2−/− and WT respectively). Moreover, we examine the interrelationship between the dispersion of repolarization time and current pulse amplitude in provoking ventricular arrhythmia.