Christoph Maack

Elevated Cytosolic Na+ Decreases Mitochondrial Ca2+ Uptake During Excitation-Contraction Coupling and Impairs Energetic Adaptation in Cardiac Myocytes

Mitochondrial Ca2+ ([Ca2+]m) regulates oxidative phosphorylation and thus contributes to energy supply and demand matching in cardiac myocytes. Mitochondria take up Ca2+ via the Ca2+ uniporter (MCU) and extrude it through the mitochondrial Na+/Ca2+ exchanger (mNCE). It is controversial whether mitochondria take up Ca2+ rapidly, on a beat-to-beat basis, or slowly, by temporally integrating cytosolic Ca2+ ([Ca2+]c) transients. Furthermore, although mitochondrial Ca2+ efflux is governed by mNCE, it is unknown whether elevated intracellular Na+ ([Na+]i) affects mitochondrial Ca2+ uptake and bioenergetics. To monitor [Ca2+]m, mitochondria of guinea pig cardiac myocytes were loaded with rhod-2–acetoxymethyl ester (rhod-2 AM), and [Ca2+]c was monitored with indo-1 after dialyzing rhod-2 out of the cytoplasm. [Ca2+]c transients, elicited by voltage-clamp depolarizations, were accompanied by fast [Ca2+]m transients, whose amplitude (Δ) correlated linearly with Δ[Ca2+]c. Under β-adrenergic stimulation, [Ca2+]m decay was ≈2.5-fold slower than that of [Ca2+]c, leading to diastolic accumulation of [Ca2+]m when amplitude or frequency of Δ[Ca2+]c increased. The MCU blocker Ru360 reduced Δ[Ca2+]m and increased Δ[Ca2+]c, whereas the mNCE inhibitor CGP-37157 potentiated diastolic [Ca2+]m accumulation. Elevating [Na+]i from 5 to 15 mmol/L accelerated mitochondrial Ca2+ decay, thus decreasing systolic and diastolic [Ca2+]m. In response to gradual or abrupt changes of workload, reduced nicotinamide-adenine dinucleotide (NADH) levels were maintained at 5 mmol/L [Na+]i, but at 15 mmol/L, the NADH pool was partially oxidized. The results indicate that (1) mitochondria take up Ca2+ rapidly and contribute to fast buffering during a [Ca2+]c transient; and (2) elevated [Na+]i impairs mitochondrial Ca2+ uptake, with consequent effects on energy supply and demand matching. The latter effect may have implications for cardiac diseases with elevated [Na+]i.

β-Adrenergic Stimulation of L-type Ca2+ Channels in Cardiac Myocytes Requires the Distal Carboxyl Terminus of α1C but Not Serine 1928

&bgr;-Adrenoceptor stimulation robustly increases cardiac L-type Ca2+ current (ICaL); yet the molecular mechanism of this effect is still not well understood. Previous reports have shown in vitro phosphorylation of a consensus protein kinase A site at serine 1928 on the carboxyl terminus of the &agr;1C subunit; however, the functional role of this site has not been investigated in cardiac myocytes. Here, we examine the effects of truncating the distal carboxyl terminus of the &agr;1C subunit at amino acid residue 1905 or mutating the putative protein kinase A site at serine 1928 to alanine in adult guinea pig myocytes, using novel dihydropyridine-insensitive &agr;1C adenoviruses, coexpressed with &bgr;2 subunits. Expression of &agr;1C truncated at 1905 dramatically attenuated the increase of peak ICaL induced by isoproterenol. However, the point mutation S1928A did not significantly attenuate the &bgr;-adrenergic response. The findings indicate that the distal carboxyl-terminus of &agr;1C plays an important role in &bgr;-adrenergic upregulation of cardiac L-type Ca2+ channels, but that phosphorylation of serine 1928 is not required for this effect.

Partial Inhibition of Sodium/Calcium Exchange Restores Cellular Calcium Handling in Canine Heart Failure

Sodium/calcium (Na+/Ca2+) exchange (NCX) overexpression is common to human heart failure and heart failure in many animal models, but its specific contribution to the cellular Ca2+ ([Ca2+]i) handling deficit is unclear. Here, we investigate the effects of exchange inhibitory peptide (XIP) on Ca2+ handling in myocytes isolated from canine tachycardic pacing-induced failing hearts. Whole-cell patch-clamped left ventricular myocytes from failing hearts (F) showed a 52% decrease in steady-state sarcoplasmic reticulum (SR) Ca2+ load and a 44% reduction in the amplitude of the [Ca2+]i transient, as compared with myocytes from normal hearts (N). Intracellular application of XIP (30 &mgr;mol/L) normalized the [Ca2+]i transient amplitude in F (3.86-fold increase), concomitant with a similar increase in SR Ca2+ load. The degree of NCX inhibition at this concentration of XIP was ≈27% and was selective for NCX: L-type Ca2+ currents and plasmalemmal Ca2+ pumps were not affected. XIP also indirectly improved the rate of [Ca2+]i removal at steady-state, secondary to Ca2+-dependent activation of SR Ca2+ uptake. The findings indicate that in the failing heart cell, NCX inhibition can improve SR Ca2+ load by shifting the balance of Ca2+ fluxes away from trans-sarcolemmal efflux toward SR accumulation. Hence, inhibition of the Ca2+ efflux mode of the exchanger could potentially be an effective therapeutic strategy for improving contractility in congestive heart failure.

Carvedilol but Not Metoprolol Reduces β-Adrenergic Responsiveness After Complete Elimination From Plasma In Vivo

Background— Carvedilol but not metoprolol exhibits persistent binding to β-adrenergic receptors (β-ARs) even after washout in cell culture experiments. Here, we determined the significance of this phenomenon on human β-ARs in vitro and in vivo. Methods and Results— Experiments were conducted on human atrial trabeculae (n=8 to 10 per group). In the presence of metoprolol, isoproterenol potency was reduced compared with controls (P <0.001). In the presence of carvedilol, isoproterenol identified 2 distinct binding sites of high (36±6%; −8.8±0.4 log mol/L) and low affinity (−6.5±0.2 log mol/L). After β-blocker washout, isoproterenol potency returned to control values in metoprolol-treated muscles, whereas in carvedilol-treated preparations, isoproterenol potency remained decreased (P <0.001 versus control). In vivo studies were performed in 9 individuals receiving metoprolol succinate (190 mg/d) or carvedilol (50 mg/d) for 11 days in a randomized crossover design. Dobutamine stress echocardiography (5 to 40 μg · kg−1 · min−1) was performed before, during, and 44 hours after application of study medication. β-Blocker medication reduced heart rate, heart rate–corrected velocity of circumferential fiber shortening, and cardiac output compared with baseline (P <0.02 to 0.0001). After withdrawal of metoprolol, all parameters returned to baseline values, whereas after carvedilol, all parameters remained reduced (P <0.05 to 0.001) despite complete plasma elimination of carvedilol. Conclusions— Carvedilol but not metoprolol inhibits the catecholamine response of the human heart beyond its plasma elimination. The persistent β-blockade by carvedilol may be explained by binding of carvedilol to an allosteric site of β-ARs.

Cardiac Sodium-Calcium Exchanger Is Regulated by Allosteric Calcium and Exchanger Inhibitory Peptide at Distinct Sites

The sarcolemmal Na+-Ca2+ exchanger (NCX) is the main Ca2+ extrusion mechanism in cardiac myocytes and is thus essential for the regulation of Ca2+ homeostasis and contractile function. A cytosolic region (f-loop) of the protein mediates regulation of NCX function by intracellular factors including inhibition by exchanger inhibitory peptide (XIP), a 20 amino acid peptide matching the sequence of an autoinhibitory region involved in allosteric regulation of NCX by intracellular Na+, Ca2+, and phosphatidylinositol-4,5-biphosphate (PIP2). Previous evidence indicates that the XIP interaction domain can be eliminated by large deletions of the f-loop that also remove activation of NCX by intracellular Ca2+. By whole-cell voltage clamping experiments, we demonstrate that deletion of residues 562–679, but not 440– 456, 498–510, or 680–685 of the f-loop selectively eliminates XIP-mediated inhibition of NCX expressed either heterologously (HEK293 and A549 cells) or in guinea pig cardiac myocytes. In contrast, by plotting INCX against reverse-mode NCX-mediated Ca2+ transients in myocytes, we demonstrate that Ca2+-dependent regulation of NCX is preserved in &Dgr;562–679, but significantly reduced in the other three deletion mutants. The findings indicate that f-loop residues 562–679 may contain the regulatory site for endogenous XIP, but this site is distinct from the Ca2+-regulatory domains of the NCX. Because regulation of the NCX by Na+ and PIP2 involves the endogenous XIP region, the &Dgr;562–679 mutant NCX may be a useful tool to investigate this regulation in the context of the whole cardiac myocyte.

β-Adrenergic Signaling in Chronic Heart Failure—Friend or Foe?

In chronic heart failure, a number of compensatory mechanisms are activated in order to maintain circulation and thus supply of the body with blood and oxygen. One important mechanism is the activation of the sympathetic nervous system, resulting in increased β-adrenergic signaling. This leads to both adaptive and pathological processes within the cell, including a desensitization of the β-adrenergic signal transduction cascade, the induction of hypertrophy, apoptosis and necrosis. It is currently a matter of debate whether a desensitization of the β-adrenergic signal transduction cascade is adaptive or maladaptive. In other words, it is not entirely clear whether in heart failure, decreased β-adrenergic signaling due to desensitization of the signaling cascade per se is a cause for cardiac dysfunction. In order to elucidate this issue, this review will focus on the consequences of increased β-adrenergic signaling, investigated by selective overexpression of distinct cascade components in mouse models. Furthermore, the impact of partial and inverse agonism of β-blockers on β-adrenergic signaling in human myocardium in vitro as well as in vivo in the clinical situation in patients with heart failure is highlighted. It is discussed whether the fact that not all β-blockers improve survival in heart failure patients may be due to their respective degree of inverse agonism.

β-Blocker treatment of chronic heart failure with special regard to Carvedilol

Congestive heart failure is a major public health problem in most Western countries. In the United States, approximately three million people suffer from heart failure, 10 % of whom are admitted to a hospital each year (57). Although in recent decades considerable changes in medical therapy have been achieved, especially by angiotensin converting enzyme (ACE) inhibitors (19, 58), mortality of heart failure still remains very high. In patients of New York Heart Association (NYHA) functional class II there is a mortality of about 10 % (2). In order to improve this poor prognosis, additional pharmacological interventions to counteract the pathophysiological changes occurring in heart failure are still important.