Sinoatrial node dysfunction induces cardiac arrhythmias in diabetic mice.

BACKGROUND: The 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. METHODS: Cardiac hemodynamic and electrophysiological characteristics were investigated in diabetic db/db and control db/+ mice. RESULTS: We 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. CONCLUSIONS: Enhanced 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.

Chronic sympathetic activation promotes downregulation of ß-adrenoceptor-mediated effects in the guinea pig heart independently of structural remodeling and systolic dysfunction.

It is uncertain if downregulation of ß-adrenoceptor signaling pathway is promoted by an enhanced adrenergic tone at an early stage of cardiac disease, or it develops secondary to detrimental local myocardial changes in advanced heart failure. We examined the integrity of ß-adrenoceptor signaling pathway upon chronic infusion of isoproterenol, a ß-adrenoceptor agonist, at a dose producing no structural left ventricular (LV) remodeling and systolic dysfunction. Subcutaneous isoproterenol infusion (400 µg kg(-1) h(-1) over 16 days) to guinea pigs using osmotic minipumps produced no change in cardiac weights, LV internal dimensions, myocyte cross-sectional area, extent of interstitial fibrosis, and basal contractile function. Isolated, perfused heart preparations from isoproterenol-treated guinea pigs exhibited attenuated responsiveness to acute ß-adrenoceptor stimulation, as evidenced by reduced LV developed pressure increase, less shortening of LV epicardial monophasic action potential and effective refractory period, and less myocardial cyclic adenosine monophosphate elevation, in response to isoproterenol exposure, when compared to saline-treated controls. Pharmacological responses to forskolin, an activator of the adenylate cyclase catalytic subunit, were well preserved in isoproterenol-treated hearts. Downregulation of ß-adrenoceptor-mediated effects upon chronic isoproterenol infusion was associated with markedly reduced stimulatory G-protein α-subunit (G(sα)) myocardial expression levels. No change in expression levels of ß-adrenoceptors, G-protein-coupled receptor kinase 2, inhibitory G-protein α-subunit (G(iα2)), and Ca(v)1.2 and K(v)7.1 ion channels was determined in isoproterenol-treated hearts. We therefore conclude that sustained adrenergic overstimulation may promote downregulation of myocardial ß-adrenoceptor-mediated effects independently of structural LV remodeling and systolic failure, an effect attributed to ß-adrenoceptor uncoupling from adenylate cyclase due to reduced G(sα)-protein expression.

Na+ channel distribution and electrophysiological heterogeneities in guinea pig ventricular wall.

We sought to explore the distribution pattern of Na(+) channels across ventricular wall, and to determine its functional correlates, in the guinea pig heart. Voltage-dependent Na(+) channel (Na(v)) protein expression levels were measured in transmural samples of ventricular tissue by Western blotting. Isolated, perfused heart preparations were used to record monophasic action potentials and volume-conducted ECG, and to measure effective refractory periods (ERPs) and pacing thresholds, in order to assess excitability, electrical restitution kinetics, and susceptibility to stimulation-evoked tachyarrhythmias at epicardial and endocardial stimulation sites. In both ventricular chambers, Na(v) protein expression was higher at endocardium than epicardium, with midmyocardial layers showing intermediate expression levels. Endocardial stimulation sites showed higher excitability, as evidenced by lower pacing thresholds during regular stimulation and downward displacement of the strength-interval curve reconstructed after extrasystolic stimulation compared with epicardium. ERP restitution assessed over a wide range of pacing rates showed greater maximal slope and faster kinetics at endocardial than epicardial stimulation sites. Flecainide, a Na(+) channel blocker, reduced the maximal ERP restitution slope, slowed restitution kinetics, and eliminated epicardial-to-endocardial difference in dynamics of electrical restitution. Greater excitability and steeper electrical restitution have been associated with greater arrhythmic susceptibility of endocardium than epicardium, as assessed by measuring ventricular fibrillation threshold, inducibility of tachyarrhythmias by rapid cardiac pacing, and the magnitude of stimulation-evoked repolarization alternans. In conclusion, higher Na(+) channel expression levels may contribute to greater excitability, steeper electrical restitution slopes and faster restitution kinetics, and greater susceptibility to stimulation-evoked tachyarrhythmias at endocardium than epicardium in the guinea pig heart.

Myocardial structural, contractile and electrophysiological changes in the guinea-pig heart failure model induced by chronic sympathetic activation.

Widely used murine models of adrenergic-induced cardiomyopathy offer little insight into electrical derangements seen in human heart failure owing to profound differences in the characteristics of ventricular repolarization in mice and rats compared with humans. We therefore sought to determine whether sustained adrenergic activation may produce a clinically relevant heart failure phenotype in the guinea-pig, an animal species whose ventricular action potential shape and restitution properties resemble those determined in humans. Isoprenaline (ISO), a ß-adrenoceptor agonist, was infused at variable dosage and duration using either subcutaneously implanted osmotic minipumps or daily injections, in an attempt to establish the relevant treatment protocol. We found that 3 months of daily ISO injections (final dose of 1 mg kg(-1), i.p.) promote heart failure evidenced by cardiac hypertrophy [increased cardiac weights, left ventricular (LV) posterior wall thickness, myocyte cross-sectional area and LV protein content], cardiac dilatation (increased LV internal diameters), basal systolic dysfunction (reduced LV fractional shortening determined by echocardiography and flattened LV systolic pressure-volume and stress-strain relationships assessed in isolated, perfused heart preparations), reduced contractile reserve in the presence of acute ß-adrenoceptor stimulation, and pulmonary oedema (increased lung weights). These changes were associated with prolongation of LV epicardial action potential, effective refractory period and QT interval, an upward shift of the electrical restitution curve determined over a wide range of diastolic intervals, and reduced maximal restitution slope. The physiological right ventricular-to-LV difference in action potential duration was eliminated in ISO-treated hearts, thereby contributing to impaired activation-to-repolarization coupling and reversed right ventricular-to-LV difference in repolarization time. In summary, we establish the guinea-pig model of ISO-induced cardiomyopathy, which enables the correlation of detrimental structural and contractile changes with repolarization abnormalities typically seen in human heart failure.

Differential effects of the transient outward K(+) current activator NS5806 in the canine left ventricle.

To examine the electrophysiological and molecular properties of the transient outward current (I(to)) in canine left ventricle using a novel I(to) activator, NS5806, I(to) was measured in isolated epicardial (Epi), midmyocardial (Mid) and endocardial (Endo) cells using whole-cell patch-clamp techniques. NS5806 activation of K(v)4.3 current was also studied in CHO-K1 cells and Xenopus laevis oocytes. In CHO-K1 cells co-transfected with K(v)4.3 and KChIP2, NS5806 (10 microM) caused a 35% increase in current amplitude and a marked slowing of current decay with tau increasing from 7.0+/-0.4 to 10.2+/-0.3 ms. In the absence of KChIP2, current decay was unaffected by NS5806. In ventricular myocytes, NS5806 increased I(to) density by 80%, 82%, and 16% in Epi, Mid, and Endo myocytes, respectively (at +40 mV) and shifted steady-state inactivation to negative potentials. NS5806 also significantly slowed decay of I(to), increasing total charge to 227%, 192% and 83% of control in Epi, Mid and Endo cells, respectively (+40 mV, p<0.05). Quantification of K(v)4.3 and KChIP2 mRNA in the 3 ventricular cell types revealed that levels of K(v)4.3 message was uniform but those of KChIP2 were significantly greater in Epi and Mid cells. The KChIP2 gradient was confirmed at the protein level by Western blot. Our results suggest that NS5806 augments I(to) by increasing current density and slowing decay and that both depend on the presence of KChIP2. I(to) and its augmentation by NS5806 are greatest in Epi and Mid cells because KChIP2 levels are highest in these cell types.

Transmural expression of ion channels and transporters in human nondiseased and end-stage failing hearts.

The cardiac action potential is primarily shaped by the orchestrated function of several different types of ion channels and transporters. One of the regional differences believed to play a major role in the progression and stability of the action potential is the transmural gradient of electrical activity across the ventricular wall. An altered balance in the ionic currents across the free wall is assumed to be a substrate for arrhythmia. A large fraction of patients with heart failure experience ventricular arrhythmia. However, the underlying substrate of these functional changes is not well-established as expression analyses of human heart failure (HF) are sparse. We have investigated steady-state RNA levels by quantitative polymerase chain reaction of ion channels, transporters, connexin 43, and miR-1 in 11 end-stage HF and seven nonfailing (NF) hearts. The quantifications were performed on endo-, mid-, and epicardium of left ventricle, enabling us to establish changes in the transmural expression gradient. Transcripts encoding Cav1.2, HCN2, Kir2.1, KCNE1, SUR1, and NCX1 were upregulated in HF compared to NF while a downregulation was observed for KChIP2, SERCA2, and miR-1. Additionally, the transmural gradient of KCNE1, KChIP2, Kir6.2, SUR1, Nav1.5, NCX1, and RyR2 found in NF was only preserved for KChiP2 and Nav1.5 in HF. The transmural gradients of NCX1, Nav1.5, and KChIP2 and the downregulation of KChIP2 were confirmed by Western blotting. In conclusion, our results reveal altered expression of several cardiac ion channels and transporters which may in part explain the increased susceptibility to arrhythmia in end-state failing hearts.

[Mutations in the mitofusin 2 gene are the most common cause of Charcot-Marie-Tooth type 2 disease]. / Mutacje w genie mitofuzyny 2 najczestsza przyczyna choroby Charcota-Mariego-Tootha typu 2.

In contrast to Charcot-Marie-Tooth type 1 disease (CMT1), which is most commonly caused by 17p11.2-p12 duplication (in 70% of CMT1 cases), the axonal form of hereditary motor and sensory neuropathy (CMT2) seemed to be a genetically heterogeneous disease group, with no single gene playing a major pathogenetic role. In 2004, 10 mutations were identified in CMT2A families in the MFN2 gene coding for the mitochondrial protein mitofusin-2, previously mapped to the 1p35-36 locus. In the last two years, MFN2 gene mutations were shown to be the most common cause of autosomal dominant hereditary axonopathy. In addition, MFN2 gene mutations were also identified in CMT type 6 (axonal neuropathy with optic nerve atrophy). Recent reports indicate that some MFN2 gene mutations may by inherited as autosomal recessive traits. As MFN2 gene mutations are the most common cause of autosomal dominant CMT2 disease (33% of cases), MFN2 gene testing may be considered a diagnostic test for CMT2.

Atrial natriuretic peptides in plasma.

Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone. These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid derivatizations. In this mini-review, we summarize measurement of the principal cardiac hormone, e.g. atrial natriuretic peptide (ANP) and its precursor fragments. We also highlight some of the analytical pitfalls and problems and the concurrent clinical "proof of concept". We conclude that biochemical research into proANP-derived peptides is still worthy of attention and that new biological insight may change our chemical perception of the markers.

KCNMA1 encoded cardiac BK channels afford protection against ischemia-reperfusion injury.

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

Contribution of Kv7 channels to basal coronary flow and active response to ischemia.

The goal of the present study was to determine the role of KCNQ-encoded Kv channels (Kv7 channels) in the passive and active regulation of coronary flow in normotensive and hypertensive rats. In left anterior descending coronary arteries from normotensive rats, structurally different Kv7.2 to 7.5 activators produced relaxations, which were considerably less in arteries from hypertensive rats and were not mimicked by the Kv7.1-specific activator R-L3. In isolated, perfused heart preparations, coronary flow rate increased in response to the Kv7.2 to 7.5 activator (S)-1 and was diminished in the presence of a Kv7 inhibitor. The expression levels of KCNQ1-5 and their known accessory KCNE1-5 subunits in coronary arteries were similar in normotensive and hypertensive rats as measured by quantitative polymerase chain reaction. However, Kv7.4 protein expression was reduced in hypertensive rats. Application of adenosine or A2A receptor agonist CGS-21680 produced concentration-dependent relaxations of coronary arteries from normotensive rats, which were attenuated by application of Kv7 inhibitors. Kv7 blockers also attenuated the ischemia-induced increase in coronary perfusion in Langendorff studies. Overall, these data establish Kv7 channels as crucial regulators of coronary flow at resting and after hypoxic insult.