Enhancing mitochondrial pyruvate metabolism ameliorates myocardial ischemic reperfusion injury.

The established clinical therapy for the treatment of acute myocardial infarction is primary percutaneous coronary intervention (PPCI) to restore blood flow to the ischemic myocardium. PPCI is effective at reperfusing the ischemic myocardium, however the rapid re-introduction of oxygenated blood also can cause ischemia-reperfusion (I/R) injury. Reperfusion injury is the culprit for up to half of the final myocardial damage, but there are no clinical interventions to reduce I/R injury. We previously demonstrated that inhibiting the lactate exporter, monocarboxylate transporter 4 (MCT4), and re-directing pyruvate towards oxidation can blunt isoproterenol-induced hypertrophy. Based on this finding, we hypothesized that the same pathway might be important during I/R. Here, we establish that the pyruvate-lactate metabolic axis plays a critical role in determining myocardial salvage following injury. Post-I/R injury, the mitochondrial pyruvate carrier (MPC), required for pyruvate oxidation, is upregulated in the surviving myocardium following I/R injury. MPC loss in cardiomyocytes caused more cell death with less myocardial salvage, which was associated with an upregulation of MCT4 in the myocardium at risk of injury. We deployed a pharmacological strategy of MCT4 inhibition with a highly selective compound (VB124) at the time of reperfusion. This strategy normalized reactive oxygen species (ROS), mitochondrial membrane potential (Δψ), and Ca 2+ , increased pyruvate entry to TCA cycle, and improved myocardial salvage and functional outcomes following I/R injury. Altogether, our data suggest that normalizing the pyruvate-lactate metabolic axis via MCT4 inhibition is a promising pharmacological strategy to mitigate I/R injury.

SMYD1a protects the heart from ischemic injury by regulating OPA1-mediated cristae remodeling and supercomplex formation.

SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.

Mitochondrial calcium uniporter stabilization preserves energetic homeostasis during Complex I impairment.

Calcium entering mitochondria potently stimulates ATP synthesis. Increases in calcium preserve energy synthesis in cardiomyopathies caused by mitochondrial dysfunction, and occur due to enhanced activity of the mitochondrial calcium uniporter channel. The signaling mechanism that mediates this compensatory increase remains unknown. Here, we find that increases in the uniporter are due to impairment in Complex I of the electron transport chain. In normal physiology, Complex I promotes uniporter degradation via an interaction with the uniporter pore-forming subunit, a process we term Complex I-induced protein turnover. When Complex I dysfunction ensues, contact with the uniporter is inhibited, preventing degradation, and leading to a build-up in functional channels. Preventing uniporter activity leads to early demise in Complex I-deficient animals. Conversely, enhancing uniporter stability rescues survival and function in Complex I deficiency. Taken together, our data identify a fundamental pathway producing compensatory increases in calcium influx during Complex I impairment.

EFHD1 ablation inhibits cardiac mitoflash activation and protects cardiomyocytes from ischemia.

Altered levels of intracellular calcium (Ca2+) are a highly prevalent feature in different forms of cardiac injury, producing changes in contractility, arrhythmias, and mitochondrial dysfunction. In cardiac ischemia-reperfusion injury, mitochondrial Ca2+ overload leads to pathological production of reactive oxygen species (ROS), activates the permeability transition, and cardiomyocyte death. Here we investigated the cardiac phenotype caused by deletion of EF-hand domain-containing protein D1 (Efhd1-/-), a Ca2+-binding mitochondrial protein whose function is poorly understood. Efhd1-/- mice are viable and have no adverse cardiac phenotypes. They feature reductions in basal ROS levels and mitoflash events, both important precursors for mitochondrial injury, though cardiac mitochondria have normal susceptibility to Ca2+ overload. Notably, we also find that Efhd1-/- mice and their cardiomyocytes are resistant to hypoxic injury.

Enhanced activity of multiple TRIC-B channels: an endoplasmic reticulum/sarcoplasmic reticulum mechanism to boost counterion currents.

KEY POINTS: There are two subtypes of trimeric intracellular cation (TRIC) channels but their distinct single-channel properties and physiological regulation have not been characterized. We examined the differences in function between native skeletal muscle sarcoplasmic reticulum (SR) K+ -channels from wild-type (WT) mice (where TRIC-A is the principal subtype) and from Tric-a knockout (KO) mice that only express TRIC-B. We find that lone SR K+ -channels from Tric-a KO mice have a lower open probability and gate more frequently in subconducting states than channels from WT mice but, unlike channels from WT mice, multiple channels gate with high open probability with a more than six-fold increase in activity when four channels are present in the bilayer. No evidence was found for a direct gating interaction between ryanodine receptor and SR K+ -channels in Tric-a KO SR, suggesting that TRIC-B-TRIC-B interactions are highly specific and may be important for meeting counterion requirements during excitation-contraction coupling in tissues where TRIC-A is sparse or absent. ABSTRACT: The trimeric intracellular cation channels, TRIC-A and TRIC-B, represent two subtypes of sarcoplasmic reticulum (SR) K+ -channel but their individual functional roles are unknown. We therefore compared the biophysical properties of SR K+ -channels derived from the skeletal muscle of wild-type (WT) or Tric-a knockout (KO) mice. Because TRIC-A is the major TRIC-subtype in skeletal muscle, WT SR will predominantly contain TRIC-A channels, whereas Tric-a KO SR will only contain TRIC-B channels. When lone SR K+ -channels were incorporated into bilayers, the open probability (Po) of channels from Tric-a KO mice was markedly lower than that of channels from WT mice; gating was characterized by shorter opening bursts and more frequent brief subconductance openings. However, unlike channels from WT mice, the Po of SR K+ -channels from Tric-a KO mice increased as increasing channel numbers were present in the bilayer, driving the channels into long sojourns in the fully open state. When co-incorporated into bilayers, ryanodine receptor channels did not directly affect the gating of SR K+ -channels, nor did the presence or absence of SR K+ -channels influence ryanodine receptor activity. We suggest that because of high expression levels in striated muscle, TRIC-A produces most of the counterion flux required during excitation-contraction coupling. TRIC-B, in contrast, is sparsely expressed in most cells and, although lone TRIC-B channels exhibit low Po, the high Po levels reached by multiple TRIC-B channels may provide a compensatory mechanism to rapidly restore K+ gradients and charge differences across the SR of tissues containing few TRIC-A channels.

Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC-A.

KEY POINTS: The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric-a gene knockout (KO) alters the single-channel function of skeletal RyR (RyR1). We find that RyR1 from Tric-a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+ , ATP, caffeine and luminal Ca2+ . In the presence of Mg2+ , ATP cannot effectively activate RyR1 from Tric-a KO mice. Additionally, RyR1 from Tric-a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of ß-adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+ -release. The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+ -release observed in skeletal muscle from Tric-a KO mice, further highlighting the importance of TRIC-A for normal physiological regulation of SR Ca2+ -release in skeletal muscle. ABSTRACT: The type A trimeric intracellular cation channel (TRIC-A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric-a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric-a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage-clamp conditions. We find that RyR1 channels from Tric-a KO mice respond normally to cytosolic Ca2+ , ATP, adenine, caffeine and to luminal Ca2+ . However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+ , ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC-A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC-A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC-A.