Background: Cardiac contractile function requires high energy from mitochondria, and Ca 2+ from the sarcoplasmic reticulum (SR). Via local Ca 2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca 2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering. Methods: We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype. Results: Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive β-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury. Conclusions: We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca 2+ /reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca 2+ signaling.

Glycogen synthase kinase 3 beta (GSK3β) is a cytosolic serine-threonine kinase first described for inhibiting glycogen synthase. GSK3β structure shows a small sequence that can work as a mitochondrial target sequence, however mitochondrial GSK3β (mtGSK3β) in the heart, and its role have not been investigated. It is known the enzyme can be activated under ischemia-reperfusion (I/R) injury and modulating mitochondrial permeability transition pore (MPTP) opening. In the nervous system, the truncation of GSK3β activates the enzyme, but in the heart, the truncation function remains unknown. We hypothesized that a small subset of GSK3β is truncated and translocated to the mitochondrial matrix, which might modulate MPTP opening . To test our hypothesis, we studied GSK3β expression by Western blot in mitochondrial fractions isolated from differential centrifugation. We found a percentage of GSK3β present in purified mitochondria (Pmito) truncated at the C-terminal side. To evaluate if mtGSK3β is involved in I/R injury, we study its expression in the fractions isolated from hearts subjected to I/R protocol. We found GSK3β is being truncated and its expression was increased in Pmito from I/R-hearts in comparison to normoxia-hearts (controls). To study the role of mtGSK3β in MPTP regulation we measure the sensitivity of the pore under a GSK3β inhibitor, SB216763 (SB21) by fluorescente of Fura-2-FF in a spectrofluorometer. MPTP Ca 2+ -sensitivity was reduced in crude mitochondria fraction (Cm, mitochondria with other organelles) and increased in Pmito under SB21 treatment. This data suggests mtGSK3β is inactive in Pmito and SB21 is inducing MPTP opening by another mechanism. Since GSK3β can be activated and inhibited by phosphorylation at T390 site (pT390) and S9 site (pS9), respectively, we measure it on mtGSK3β. We found pT390 is not present in Pmito likely missing during truncation, while pS9 is in a low amount. Moreover, pS9 increases on Pmito from hearts subjected to I/R protocol. All the data together showed that a small percentage of GSK3β is truncated and translocated within the mitochondrial matrix. However, this mtGSK3β is not involved in the modulation of MPTP opening, since the lack of a C-terminal side causes its inactivation.

The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, maintenance of higher energy expenditure during the fast and reduced binge-eating during the feeding period. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

The mitochondrial permeability transition pore (mPTP) plays a critical role in the pathogenesis of cardiovascular diseases, including ischemia/reperfusion injury. Although the pore structure is still unresolved, the mechanism through which cyclophilin D (CypD) regulates mPTP opening is the subject of intensive studies. While post-translational modifications of CypD have been shown to modulate pore opening, specific phosphorylation sites of CypD have not yet been identified. We hypothesized here that phosphorylation of CypD on a serine residue controls mPTP opening and subsequent cell death at reperfusion. We combined in silico analysis with in vitro and genetic manipulations to determine potential CypD phosphorylation sites and their effect on mitochondrial function and cell death. Importantly, we developed an in vivo intramyocardial adenoviral strategy to assess the effect of the CypD phosphorylation event on infarct size. Our results show that although CypD can potentially be phosphorylated at multiple serine residues, only the phosphorylation status at S191 directly impacts the ability of CypD to regulate the mPTP. Protein-protein interaction strategies showed that the interaction between CypD and oligomycin sensitivity-conferring protein (OSCP) was reduced by 45% in the phosphoresistant S191A mutant, whereas it was increased by 48% in the phosphomimetic S191E mutant cells. As a result, the phosphoresistant CypD S191A mutant was protected against 18 h starvation whereas cell death was significantly increased in phosphomimetic S191E group, associated with mitochondrial respiration alteration and ROS production. As in vivo proof of concept, in S191A phosphoresistant rescued CypD-KO mice developed significantly smaller infarct as compared to WT whereas infarct size was drastically increased in S191E phosphomimetic rescued mice. We conclude that CypD phosphorylation at S191 residue leads to its binding to OSCP and thus sensitizes mPTP opening for the subsequent cell death.

Neginskaya et al. discuss the very low number of calcium-induced permeability transition pores in the single mitochondrion.

The mitochondrial matrix ATPase associated with diverse cellular activities (m-AAA) protease spastic paraplegia 7 (SPG7) has been recently implicated as either a negative or positive regulatory component of the mitochondrial permeability transition pore (mPTP) by two research groups. To address this controversy, we investigated possible mechanisms that explain the discrepancies between these two studies. We found that loss of the SPG7 gene increased resistance to Ca2+-induced mPTP opening. However, this occurs independently of cyclophilin D (cyclosporine A insensitive) rather it is through decreased mitochondrial Ca2+ concentrations and subsequent adaptations mediated by impaired formation of functional mitochondrial Ca2+ uniporter complexes. We found that SPG7 directs the m-AAA complex to favor association with the mitochondrial Ca2+ uniporter (MCU) and MCU processing regulates higher order MCU-complex formation. The results suggest that SPG7 does not constitute a core component of the mPTP but can modulate mPTP through regulation of the basal mitochondrial Ca2+ concentration.

Mitofusin 2 (MFN2), originally recognized for mediating mitochondrial fusion, has been also established as a major player in the endoplasmic reticulum (ER) and mitochondria interaction. MFN2 ablation reduced the efficacy of interorganelle Ca2+ signalling, which effect has been largely attributed to MFN2's ER- mitochondrial tethering role. However, the mechanism by which MFN2 regulates Ca2+ homeostasis between the organelles remains unclear. Here, we introduced a non-canonical role of MFN2 in ER Ca2+ regulation potentially via an interaction with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). To study the effect of MFN2-overexpression (OE) on ER Ca2+ homeostasis two sets of Myc-tagged MFN2 plasmids were used: wild-type present in both ER and mitochondria (WT-MFN2) and MFN2 lacking the mitochondrial targeting sequence (ER-MFN2). Western blotting and confocal microscopy confirmed OE and localization of tagged MFN2. Effect of MFN2 knock-out (KO) and WT-, ER-MFN2-OE on Ca2+ dynamics was studied in permeabilized HEK293T and MEF cells. [Ca2+] in the ER lumen ([Ca2+]ER) was visualized using GEM-CEPIA1er Ca2+-biosensor. Basal [Ca2+]ER was significantly lower in WT- and ER-MFN2-OE cells vs control and MFN2-KO cells where we observed highest level of the basal [Ca2+]ER. Importantly, the rate of [Ca2+]ER re-uptake by SERCA after its depletion by reversible inhibitor cyclopiazonic acid was significantly faster in MFN2-KO cells vs control. In WT- and ER-MFN2-OE [Ca2+]ER re-uptake was significantly slower than in control with more dramatic effect in ER-MFN2-OE. Initial co-immunoprecipitation and FRET experiments suggest SERCA interaction with MFN2. Thus, our data shows that in addition to the ER-mito tethering role, MFN2 might interact with and inhibit SERCA, and potentially regulate ER-mito calcium cross-talk via changing ER Ca2+ handling.

Mitochondrial Ca2+ uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca2+ uptake and our current understanding of mitochondrial Ca2+ homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca2+ uniporter complex.

Aims: Mitochondrial Ca homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated a1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. Results: a1-adrenoceptor (a1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that a1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca overload. Innovation: Our data indicate that inhibition of a1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. Conclusion: The a1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca entry and apoptosis in cardiac cells. Antioxid. Redox Signal. 00, 000–000.

Introduction Mitochondrial Ca 2+ uptake via the mitochondrial Ca 2+ uniporter (MCU) is critical for the cardiomyocyte survival and death. Basal tyrosine phosphorylation (P‐Y) of MCU was reported from mass spectroscopy of human tissues. However, it is still unclear whether the post‐translational modifications of the MCU (e.g. phosphorylation) modulates its channel properties as well as mitochondrial and cellular functions in cardiomyocytes. Hypothesis P‐Y of MCU activates mitochondrial Ca 2+ uptake and induces mitochondrial Ca 2+ overload and cardiomyocyte death. Methods Non‐phospho‐mimetic MCUs (MCU‐YFs; changed from Y to F) were generated by PCR‐based site direct mutagenesis. Mitochondrial Ca 2+ concentration was measured by matrix‐targeted Ca 2+ biosensors using confocal microscopy. Results a 1 ‐adrenoceptor (a 1 ‐AR) signaling activated mitochondrial matrix‐localized proline‐rich tyrosine kinase 2 (Pyk2) and enhanced the interaction between Pyk2 and MCU, which subsequently increased P‐Y of MCU and mitochondrial Ca 2+ uptake in rat cardiomyocytes. We confirmed that two tyrosine sites showed an increase of phosphorylation levels in response to a 1 ‐AR stimulation in situ ( Fig. A ). In addition, the overexpression of these MCU‐YFs failed to increase mitochondrial Ca 2+ uptake in response to cytosolic Ca 2+ elevation, whereas wild‐type MCU overexpression dramatically accelerated mitochondrial Ca 2+ uptake compared to control cells ( Fig. B ). Moreover, persistent a 1 ‐AR stimulation increased mitochondrial reactive oxygen species (mROS) generation, cytochrome C release, and cardiomyocyte death. These effects were abolished by the overexpression of a dominant‐negative mutant of MCU in cardiomyocytes. Conclusion MCU contains Pyk2‐specific phosphorylation site(s) and P‐Y of MCU activates its channel function. Persistent P‐Y of MCU induces mitochondrial Ca 2+ overload, mROS generation and apoptosis in cardiomyocytes. Support or Funding Information This work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).

Heart failure (HF) occurs in response to various types of stimulus including Gq‐protein coupled receptor (GqPCR) stimulation. GqPCR‐mediated mitochondrial dysfunction is frequently observed in HF animal models, but the molecular mechanism remains unclear. Recently, Protein kinase D (PKD) located at GqPCR downstream has been recognized as a key signaling nodal point affecting various cardiac functions. Therefore, we hypothesize that PKD acts as a mitochondrial signaling component and induces mitochondrial dysfunctions under GqPCR‐mediated HF. Using the outer mitochondrial membrane (OMM)‐targeted CFP (mt‐CFP), we found that the value of the Förster resonance energy transfer (FRET) between mt‐CFP and YFP‐tagged PKD significantly increases upon GqPCR stimulation, indicating PKD translocation from cytosol to the OMM. Next, using the FRET‐based OMM‐targeted PKD‐activity sensor, we observed that PKD is activated at the OMM after GqPCR stimulation. Furthermore, we found that GqPCR‐mediated PKD activation at the OMM induced mitochondrial fragmentation, increased reactive oxygen species generation and mitochondrial permeability transition pore opening, followed by caspase‐3 activation in cardiomyocytes. These morphological and functional alterations in cardiac mitochondria were mediated via PKD‐dependent phosphorylation of Dynamin‐Like Protein 1 (DLP1) at S637. Importantly, PKD‐dependent DLP1 phosphorylation concurrent with abnormal mitochondrial morphology and apoptotic signaling were also observed in ventricular tissue from transgenic mice with cardiac‐specific overexpression of constitutively active Gaq. In summary, we conclude that GqPCR stimulation induces PKD translocation to the OMM and induces mitochondrial fragmentation and dysfunction, which likely contributes to cardiomyocyte dysfunction during HF.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).

The mitochondrial permeability transition pore was originally described in the 1970’s as a Ca2+ activated pore and has since been attributed to the pathogenesis of many diseases. Here we evaluate how each of the current models of the pore complex fit to what is known about how Ca2+ regulates the pore, and any insight that provides into the molecular identity of the pore complex. We also discuss the central role of Ca2+ in modulating the pore’s open probability by directly regulating processes, such as ATP/ADP balance through the tricarboxylic acid cycle, electron transport chain, and mitochondrial membrane potential. We review how Ca2+ influences second messengers such as reactive oxygen/nitrogen species production and polyphosphate formation. We discuss the evidence for how Ca2+ regulates post-translational modification of cyclophilin D including phosphorylation by glycogen synthase kinase 3 beta, deacetylation by sirtuins, and oxidation/ nitrosylation of key residues. Lastly we introduce a novel view into how Ca2+ activated proteolysis through calpains in the mitochondria may be a driver of sustained pore opening during pathologies such as ischemia reperfusion injury.

Human pedigrees carrying the ryanodine receptor type 1 (RyR1) mutations frequently show skeletal muscle (SM) disorders including malignant hyperthermia (MH). There are also case reports describing sudden cardiac death (SCD) in MH patients in the conscious condition without anesthesia. These observations cannot be explained as a secondary effect of their SM dysfunction; however, the molecular mechanism underlying cardiac phenotypes in MH is still unknown. We previously reported that a low level of RyR1 is expressed in the mitochondria (termed as mitochondrial RyR1: mRyR1), but not in the sarcoplasmic reticulum (SR) in the cardiomyocytes and serves as an important mitochondrial Ca2+ influx pathway in addition to the mitochondrial Ca2+ uniporter in cardiomyocytes. We also reported, using knock‐in mice carrying a MH‐related RyR1 mutation Y522S (YS) that YS hearts exhibit disrupted mitochondrial morphology as well as compromised mitochondrial functions with a high cellular oxidative state, which can account for cardiac phenotypes in MH. In addition, YS heart developed multiple ventricular extrasystoles by β‐adrenergic stimulation. Therefore, we hypothesize that YS‐RyR1s form “leaky channel” at mitochondria and induce mitochondrial Ca2+ overload, which alters the cellular Ca2+ handling in cardiomyocyte. Using isolated mitochondria or isolated cardiomyocytes under confocal microscope, we found that YS cardiomyocytes have higher basal mitochondrial Ca2+ concentration, depolarized mitochondrial membrane potential and slower cytosolic Ca2+ clearance as compared to WT. Moreover, pretreatment of RyR1 blocker dantrolene prevented these changes in YS cardiomyocytes and normalized their Ca2+ handling profiles similar to those in WT. In conclusion, these results indicate that chronic mitochondrial Ca2+ overload via leaky mutant mRyR1 damages cardiac mitochondrial functions/structures, reduces cytosolic Ca2+ buffering capacity and induces cellular oxidation, which may increase arrhythmogenic events in MH.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).

Cardiac fibroblasts (CFs) are the most prevalent cell type in the heart in addition to cardiomyocytes and play key roles in regulating myocardial physiological function and pathophysiological remodeling. Clinical observations and basic research data strongly suggest that CFs can respond to various stimuli including, angiotensin II (AT‐II), the levels of which are increased in the remodeling heart and participates in remodeling of the failing heart by AT‐II‐mediated pathological CF proliferation. It has been shown that CF proliferation may occur via enhanced production of reactive oxygen species (ROS), but the detailed signal transduction remains unclear. We previously reported that the enhancement of mitochondrial Ca2+ uptake by mitochondrial Ca2+ uniporter (MCU) induces mitochondrial superoxide (mtSO) generation in cardiomyocytes. Therefore, we hypothesize that Ang‐II stimulation enhances mitochondrial Ca2+‐ induced mtSO generation in CFs, which can activate ROS‐dependent proliferation signaling in CFs. First, using a mitochondria‐targeted Ca2+ biosensor, we found that AT‐II (≥1 μM) stimulation induces significant mitochondrial Ca2+ uptake in response to the Ca2+ release from the endoplasmic reticulum in neonatal rat CFs (NCF). In addition, AT‐II stimulation increases the mtSO levels detected by a mtSO indicator MitoSOX Red. These effects were significantly blocked by the pretreatment of an AT‐receptor antagonist losartan. Next, we confirmed that AT‐II application activates proliferative pathway, including ERK1/2, p38 and JNK1/2 in time‐dependent manner, which was abolished by losartan pretreatment. Lastly, pretreatment of a mitochondria‐targeted antioxidant, Mito‐tempo also significantly inhibited AT‐II‐mediated activation of the proliferative pathway without changing the AT‐II‐induced the mitochondrial Ca2+ uptake profile. Our results indicate that mtSO generation induced by mitochondrial Ca2+ accumulation works as a stimulator of the Ang II–induced proliferative pathway in cardiac fibroblasts.Support or Funding InformationThis work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).

The mitochondrial permeability transition pore was originally described in the 1970’s as a Ca 2+ activated pore and has since been attributed to the pathogenesis of many diseases. Here we evaluate how each of the current models of the pore complex fit to what is known about how Ca 2+ regulates the pore, and any insight that provides into the molecular identity of the pore complex. We also discuss the central role of Ca 2+ in modulating the pore’s open probability by directly regulating processes, such as ATP/ADP balance through the tricarboxylic acid cycle, electron transport chain, and mitochondrial membrane potential. We review how Ca 2+ influences second messengers such as reactive oxygen/nitrogen species production and polyphosphate formation. We discuss the evidence for how Ca 2+ regulates post-translational modification of cyclophilin D including phosphorylation by glycogen synthase kinase 3 beta, deacetylation by sirtuins, and oxidation/ nitrosylation of key residues. Lastly we introduce a novel view into how Ca 2+ activated proteolysis through calpains in the mitochondria may be a driver of sustained pore opening during pathologies such as ischemia reperfusion injury.