Fueling the heartbeat: Dynamic regulation of intracellular ATP during excitation-contraction coupling in ventricular myocytes.

The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca2+ handling, and actin-myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP]i) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP]i in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP]i. Notably, diastolic [ATP]i was <1 mM, which is eightfold to 10-fold lower than previously estimated. Accordingly, ATP-sensitive K+ (KATP) channels were active at physiological [ATP]i. Cells exhibited two distinct types of ATP fluctuations during an action potential: net increases (Mode 1) or decreases (Mode 2) in [ATP]i. Mode 1 [ATP]i increases necessitated Ca2+ entry and release from the sarcoplasmic reticulum (SR) and were associated with increases in mitochondrial Ca2+. By contrast, decreases in mitochondrial Ca2+ accompanied Mode 2 [ATP]i decreases. Down-regulation of the protein mitofusin 2 reduced the magnitude of [ATP]i fluctuations, indicating that SR-mitochondrial coupling plays a crucial role in the dynamic control of ATP levels. Activation of ß-adrenergic receptors decreased [ATP]i, underscoring the energetic impact of this signaling pathway. Finally, our work suggests that cross-bridge cycling is the largest consumer of ATP in a ventricular myocyte during an action potential. These findings provide insights into the energetic demands of EC coupling and highlight the dynamic nature of ATP concentrations in cardiac muscle.

Calcium Channelopathies: Structural Insights into Disorders of the Muscle Excitation-Contraction Complex.

Ion channels are membrane proteins responsible for the passage of ions down their electrochemical gradients and across biological membranes. In this, they generate and shape action potentials and provide secondary messengers for various signaling pathways. They are often part of larger complexes containing auxiliary subunits and regulatory proteins. Channelopathies arise from mutations in the genes encoding ion channels or their associated proteins. Recent advances in cryo-electron microscopy have resulted in an explosion of ion channel structures in multiple states, generating a wealth of new information on channelopathies. Disease-associated mutations fall into different categories, interfering with ion permeation, protein folding, voltage sensing, ligand and protein binding, and allosteric modulation of channel gating. Prime examples of these are Ca2+-selective channels expressed in myocytes, for which multiple structures in distinct conformational states have recently been uncovered. We discuss the latest insights into these calcium channelopathies from a structural viewpoint.

Acute phosphatidylinositol 4,5 bisphosphate depletion destabilizes sarcolemmal expression of cardiac L-type Ca2+ channel CaV1.2.

CaV1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility.

The distal C terminus of the dihydropyridine receptor ß1a subunit is essential for tetrad formation in skeletal muscle.

The skeletal muscle dihydropyridine receptor (DHPR) ß1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation­contraction (EC) coupling. Previously, we showed that all four vertebrate ß isoforms (ß1­ß4) facilitate α1S triad targeting and, except for ß3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488­7493 (2013)]. Consequently, ß3 failed to restore EC coupling despite the fact that both ß3 and ß1a restore tetrads. Thus, all ß-subunits are able to restore triad targeting, but only ß1a restores both tetrads and proper DHPR­RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488­7493 (2013)]. To investigate the molecular region(s) of ß1a responsible for the tetradic arrangement of DHPRs and thus DHPR­RyR1 coupling, we expressed loss- and gain-of-function chimeras between ß1a and ß4, with systematically swapped domains in zebrafish strain relaxed (ß1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. ß1a/ß4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from ß4 showed complete restoration of SR Ca2+ release. However, chimera ß1a/ß4(C) with ß4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera ß4/ß1a(C) with ß1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of ß1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR­RyR1 interaction, essential for skeletal muscle EC coupling.

A molecular complex of Cav1.2/CaMKK2/CaMK1a in caveolae is responsible for vascular remodeling via excitation-transcription coupling.

Elevation of intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitation­transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.

Dual mode of action of IP3-dependent SR-Ca2+ release on local and global SR-Ca2+ release in ventricular cardiomyocytes.

In heart muscle, the physiological function of IP3-induced Ca2+ release (IP3ICR) from the sarcoplasmic reticulum (SR) is still the subject of intense study. A role of IP3ICR may reside in modulating Ca2+-dependent cardiac arrhythmogenicity. Here we observe the propensity of spontaneous intracellular Ca2+ waves (SCaW) driven by Ca2+-induced Ca2+ release (CICR) in ventricular myocytes as a correlate of arrhythmogenicity on the organ level. We observe a dual mode of action of IP3ICR on SCaW generation in an IP3R overexpression model. This model shows a mild cardiac phenotype and mimics pathophysiological conditions of increased IP3R activity. In this model, IP3ICR was able to increase or decrease the occurrence of SCaW depending on global Ca2+ activity. This IP3ICR-based regulatory mechanism can operate in two "modes" depending on the intracellular CICR activity and efficiency (e.g. SCaW and/or local Ryanodine Receptor (RyR) Ca2+ release events, respectively): a) in a mode that augments the CICR mechanism at the cellular level, resulting in improved excitation-contraction coupling (ECC) and ultimately better contraction of the myocardium, and b) in a protective mode in which the CICR activity is curtailed to prevent the occurrence of Ca2+ waves at the cellular level and thus reduce the probability of arrhythmogenicity at the organ level.

The Contractile Machines of the Heart.

This chapter will describe basic structural and functional features of the contractile apparatus of muscle cells of the heart, namely, cardiomyocytes and smooth muscle cells. Cardiomyocytes form the contractile myocardium of the heart, while smooth muscle cells form the contractile coronary vessels. Both muscle types have distinct properties and will be considered with respect to their cellular appearance (brick-like cross-striated versus spindle-like smooth), arrangement of contractile proteins (sarcomeric versus non-sarcomeric organization), calcium activation mechanisms (thin-filament versus thick-filament regulation), contractile features (fast and phasic versus slow and tonic), energy metabolism (high oxygen versus low oxygen demand), molecular motors (type II myosin isoenzymes with high adenosine diphosphate [ADP]-release rate versus myosin isoenzymes with low ADP-release rates), chemomechanical energy conversion (high adenosine triphosphate [ATP] consumption and short duty ratio versus low ATP consumption and high duty ratio of myosin II cross-bridges [XBs]), and excitation-contraction coupling (calcium-induced calcium release versus pharmacomechanical coupling). Part of the work has been published (Neuroscience - From Molecules to Behavior", Chap. 22, Galizia and Lledo eds 2013, Springer-Verlag; with kind permission from Springer Science + Business Media).

Voltage sensor movements of CaV1.1 during an action potential in skeletal muscle fibers.

The skeletal muscle L-type Ca2+ channel (CaV1.1) works primarily as a voltage sensor for skeletal muscle action potential (AP)-evoked Ca2+ release. CaV1.1 contains four distinct voltage-sensing domains (VSDs), yet the contribution of each VSD to AP-evoked Ca2+ release remains unknown. To investigate the role of VSDs in excitation-contraction coupling (ECC), we encoded cysteine substitutions on each S4 voltage-sensing segment of CaV1.1, expressed each construct via in vivo gene transfer electroporation, and used in cellulo AP fluorometry to track the movement of each CaV1.1 VSD in skeletal muscle fibers. We first provide electrical measurements of CaV1.1 voltage sensor charge movement in response to an AP waveform. Then we characterize the fluorescently labeled channels' VSD fluorescence signal responses to an AP and compare them with the waveforms of the electrically measured charge movement, the optically measured free myoplasmic Ca2+, and the calculated rate of Ca2+ release from the sarcoplasmic reticulum for an AP, the physiological signal for skeletal muscle fiber activation. A considerable fraction of the fluorescence signal for each VSD occurred after the time of peak Ca2+ release, and even more occurred after the earlier peak of electrically measured charge movement during an AP, and thus could not directly reflect activation of Ca2+ release or charge movement, respectively. However, a sizable fraction of the fluorometric signals for VSDs I, II, and IV, but not VSDIII, overlap the rising phase of charge moved, and even more for Ca2+ release, and thus could be involved in voltage sensor rearrangements or Ca2+ release activation.

Transcriptional regulation of intermolecular Ca2+ signaling in hibernating ground squirrel cardiomyocytes: The myocardin-junctophilin axis.

The contraction of heart cells is controlled by the intermolecular signaling between L-type Ca2+ channels (LCCs) and ryanodine receptors (RyRs), and the nanodistance between them depends on the interaction between junctophilin-2 (JPH2) in the sarcoplasmic reticulum (SR) and caveolin-3 (CAV3) in the transversal tubule (TT). In heart failure, decreased expression of JPH2 compromises LCC-RyR communication leading to deficient blood-pumping power. In the present study, we found that JPH2 and CAV3 transcription was concurrently regulated by serum response factor (SRF) and myocardin. In cardiomyocytes from torpid ground squirrels, compared with those from euthermic counterparts, myocardin expression was up-regulated, which boosted both JPH2 and CAV3 expression. Transmission electron microscopic imaging showed that the physical coupling between TTs and SRs was tightened during hibernation and after myocardin overexpression. Confocal Ca2+ imaging under the whole-cell patch clamp condition revealed that these changes enhanced the efficiency of LCC-RyR intermolecular signaling and fully compensated the adaptive down-regulation of LCCs, maintaining the power of heart contraction while avoiding the risk of calcium overload during hibernation. Our finding not only revealed an essential molecular mechanism underlying the survival of hibernating mammals, but also demonstrated a "reverse model of heart failure" at the molecular level, suggesting a strategy for treating heart diseases.

Ca2+ spark latency and control of intrinsic Ca2+ release dyssynchrony in rat cardiac ventricular muscle cells.

Cardiac excitation-contraction coupling (ECC) depends on Ca2+ release from intracellular stores via ryanodine receptors (RyRs) triggered by L-type Ca2+ channels (LCCs). Uncertain numbers of RyRs and LCCs form 'couplons' whose activation produces Ca2+ sparks, which summate to form a cell-wide Ca2+ transient that switches on contraction. Voltage (Vm) changes during the action potential (AP) and stochasticity in channel gating should create variability in Ca2+ spark timing, but Ca2+ transient wavefronts have remarkable uniformity. To examine how this is achieved, we measured the Vm-dependence of evoked Ca2+ spark probability (Pspark) and latency over a wide voltage range in rat ventricular cells. With depolarising steps, Ca2+ spark latency showed a U-shaped Vm-dependence, while repolarising steps from 50 mV produced Ca2+ spark latencies that increased monotonically with Vm. A computer model based on reported channel gating and geometry reproduced our experimental data and revealed a likely RyR:LCC stoichiometry of âˆ¼ 5:1 for the Ca2+ spark initiating complex (IC). Using the experimental AP waveform, the model revealed a high coupling fidelity (Pcpl âˆ¼ 0.5) between each LCC opening and IC activation. The presence of âˆ¼ 4 ICs per couplon reduced Ca2+ spark latency and increased Pspark to match experimental data. Variability in AP release timing is less than that seen with voltage steps because the AP overshoot and later repolarization decrease Pspark due to effects on LCC flux and LCC deactivation respectively. This work provides a framework for explaining the Vm- and time-dependence of Pspark, and indicates how ion channel dispersion in disease can contribute to dyssynchrony in Ca2+ release.

Membrane remodelling triggers maturation of excitation-contraction coupling in 3D-shaped human-induced pluripotent stem cell-derived cardiomyocytes.

The prospective use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) for cardiac regenerative medicine strongly depends on the electro-mechanical properties of these cells, especially regarding the Ca2+-dependent excitation-contraction (EC) coupling mechanism. Currently, the immature structural and functional features of hiPSC-CM limit the progression towards clinical applications. Here, we show that a specific microarchitecture is essential for functional maturation of hiPSC-CM. Structural remodelling towards a cuboid cell shape and induction of BIN1, a facilitator of membrane invaginations, lead to transverse (t)-tubule-like structures. This transformation brings two Ca2+ channels critical for EC coupling in close proximity, the L-type Ca2+ channel at the sarcolemma and the ryanodine receptor at the sarcoplasmic reticulum. Consequently, the Ca2+-dependent functional interaction of these channels becomes more efficient, leading to improved spatio-temporal synchronisation of Ca2+ transients and higher EC coupling gain. Thus, functional maturation of hiPSC-cardiomyocytes by optimised cell microarchitecture needs to be considered for future cardiac regenerative approaches.

Beat-by-Beat Cardiomyocyte T-Tubule Deformation Drives Tubular Content Exchange.

RATIONALE: The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates. OBJECTIVE: We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid. METHODS AND RESULTS: Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics. CONCLUSIONS: Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.

PDE1 Inhibition Modulates Cav1.2 Channel to Stimulate Cardiomyocyte Contraction.

[Figure: see text].

CaMKII Serine 280 O-GlcNAcylation Links Diabetic Hyperglycemia to Proarrhythmia.

[Figure: see text].

Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca2+ Dynamics.

RATIONALE: Ca2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. OBJECTIVE: To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. METHODS AND RESULTS: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after ß-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. CONCLUSIONS: The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

RYR2-ryanodinopathies: from calcium overload to calcium deficiency.

The sarcoplasmatic reticulum (SR) cardiac ryanodine receptor/calcium release channel RyR2 is an essential regulator of cardiac excitation-contraction coupling and intracellular calcium homeostasis. Mutations of the RYR2 are the cause of rare, potentially lethal inherited arrhythmia disorders. Catecholaminergic polymorphic ventricular tachycardia (CPVT) was first described more than 20 years ago and is the most common and most extensively studied cardiac ryanodinopathy. Over time, other distinct inherited arrhythmia syndromes have been related to abnormal RyR2 function. In addition to CPVT, there are at least two other distinct RYR2-ryanodinopathies that differ mechanistically and phenotypically from CPVT: RYR2 exon-3 deletion syndrome and the recently identified calcium release deficiency syndrome (CRDS). The pathophysiology of the different cardiac ryanodinopathies is characterized by complex mechanisms resulting in excessive spontaneous SR calcium release or SR calcium release deficiency. While the vast majority of CPVT cases are related to gain-of-function variants of the RyR2 protein, the recently identified CRDS is linked to RyR2 loss-of-function variants. The increasing number of these cardiac 'ryanodinopathies' reflects the complexity of RYR2-related cardiogenetic disorders and represents an ongoing challenge for clinicians. This state-of-the-art review summarizes our contemporary understanding of RYR2-related inherited arrhythmia disorders and provides a systematic and comprehensive description of the distinct cardiac ryanodinopathies discussing clinical aspects and molecular insights. Accurate identification of the underlying type of cardiac ryanodinopathy is essential for the clinical management of affected patients and their families.

CaV1.1 Calcium Channel Signaling Complexes in Excitation-Contraction Coupling: Insights from Channelopathies.

In skeletal muscle, excitation-contraction (EC) coupling relies on the mechanical coupling between two ion channels: the L-type voltage-gated calcium channel (CaV1.1), located in the sarcolemma and functioning as the voltage sensor of EC coupling, and the ryanodine receptor 1 (RyR1), located on the sarcoplasmic reticulum serving as the calcium release channel. To this day, the molecular mechanism by which these two ion channels are linked remains elusive. However, recently, skeletal muscle EC coupling could be reconstituted in heterologous cells, revealing that only four proteins are essential for this process: CaV1.1, RyR1, and the cytosolic proteins CaVß1a and STAC3. Due to the crucial role of these proteins in skeletal muscle EC coupling, any mutation that affects any one of these proteins can have devastating consequences, resulting in congenital myopathies and other pathologies.Here, we summarize the current knowledge concerning these four essential proteins and discuss the pathophysiology of the CaV1.1, RyR1, and STAC3-related skeletal muscle diseases with an emphasis on the molecular mechanisms. Being part of the same signalosome, mutations in different proteins often result in congenital myopathies with similar symptoms or even in the same disease.

[Mechano-energetic defects in heart failure]. / Mechanoenergetische Defekte bei Herzinsuffizienz.

Heart failure is characterized by defects in excitation-contraction coupling, energetic deficit and oxidative stress. The energy for cardiac contraction and relaxation is provided in mitochondria, whose function is tightly regulated by excitation-contraction coupling in cardiac myocytes. In heart failure with reduced ejection fraction (HFrEF), alterations in the ion balance in cardiac myocytes impair mitochondrial Ca2+ uptake, which is required for activation of the Krebs cycle, causing an energetic deficit and oxidative stress in mitochondria. Recent clinical studies suggest that in heart failure with preserved ejection fraction (HFpEF), in stark contrast to HFrEF, hypercontractility often occurs as an attempt to compensate for a pathological increase in systemic and pulmonary vascular resistance. This hypercontractility increases cardiac energy and oxygen demands at rest and reduces the contractile, diastolic and coronary reserves, preventing an adequate increase in cardiac output during exercise. Moreover, increased contractility causes long-term maladaptive remodeling processes due to oxidative stress and redox-sensitive prohypertrophic signaling pathways. As overweight and diabetes, particularly in the interplay with hemodynamic stress, are important risk factors for the development of HFpEF, interventions targeting metabolism in particular could ameliorate the development and progression of HFpEF.

Arrhythmogenic late Ca2+ sparks in failing heart cells and their control by action potential configuration.

Sudden death in heart failure patients is a major clinical problem worldwide, but it is unclear how arrhythmogenic early afterdepolarizations (EADs) are triggered in failing heart cells. To examine EAD initiation, high-sensitivity intracellular Ca2+ measurements were combined with action potential voltage clamp techniques in a physiologically relevant heart failure model. In failing cells, the loss of Ca2+ release synchrony at the start of the action potential leads to an increase in number of microscopic intracellular Ca2+ release events ("late" Ca2+ sparks) during phase 2-3 of the action potential. These late Ca2+ sparks prolong the Ca2+ transient that activates contraction and can trigger propagating microscopic Ca2+ ripples, larger macroscopic Ca2+ waves, and EADs. Modification of the action potential to include steps to different potentials revealed the amount of current generated by these late Ca2+ sparks and their (subsequent) spatiotemporal summation into Ca2+ ripples/waves. Comparison of this current to the net current that causes action potential repolarization shows that late Ca2+ sparks provide a mechanism for EAD initiation. Computer simulations confirmed that this forms the basis of a strong oscillatory positive feedback system that can act in parallel with other purely voltage-dependent ionic mechanisms for EAD initiation. In failing heart cells, restoration of the action potential to a nonfailing phase 1 configuration improved the synchrony of excitation-contraction coupling, increased Ca2+ transient amplitude, and suppressed late Ca2+ sparks. Therapeutic control of late Ca2+ spark activity may provide an additional approach for treating heart failure and reduce the risk for sudden cardiac death.

BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor.

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.