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Background We reported 3 novel nonsynonymous single nucleotide variants of Bcl2-associated athanogene 3 (BAG3) in African Americans with heart failure (HF) that are associated with a 2-fold increase in cardiac events (HF hospitalization, heart transplantation, or death). Methods and Results We expressed BAG3 variants (P63A, P380S, and A479V) via adenovirus-mediated gene transfer in adult left ventricular myocytes isolated from either wild-type (WT) or cardiac-specific BAG3 haploinsufficient (cBAG3+/−) mice: the latter to simulate the clinical situation in which BAG3 variants are only found on 1 allele. Compared with WT myocytes, cBAG3+/− myocytes expressed approximately 50% of endogenous BAG3 levels and exhibited decreased [Ca2+]i and contraction amplitudes after isoproterenol owing to decreased L-type Ca2+ current. BAG3 repletion with WT BAG3 but not P380S, A479V, or P63A/P380S variants restored contraction amplitudes in cBAG3+/− myocytes to those measured in WT myocytes, suggesting excitation–contraction abnormalities partly account for HF in patients harboring these mutants. Because P63A is near the WW domain (residues 21–55) and A479V is in the BAG domain (residues 420–499), we expressed BAG3 deletion mutants (Δ1–61 and Δ421–575) in WT myocytes and demonstrated that the BAG but not the WW domain was involved in enhancement of excitation–contraction by isoproterenol. Conclusions The BAG3 variants contribute to HF in African American patients partly by decreasing myocyte excitation–contraction under stress, and that both the BAG and PXXP domains are involved in mediating β-adrenergic responsiveness in myocytes.

Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in maintaining cell survival following oxidant injury. Here, we show that TRPM2 is highly expressed in acute myeloid leukemia (AML). The role of TRPM2 in AML was studied following depletion with CRISPR/Cas9 technology in U937 cells. In in vitro experiments and in xenografts, depletion of TRPM2 in AML inhibited leukemia proliferation, and doxorubicin sensitivity was increased. Mitochondrial function including oxygen consumption rate and ATP production was reduced, impairing cellular bioenergetics. Mitochondrial membrane potential and mitochondrial calcium uptake were significantly decreased in depleted cells. Mitochondrial reactive oxygen species (ROS) were significantly increased, and Nrf2 was decreased, reducing the antioxidant response. In TRPM2-depleted cells, ULK1, Atg7, and Atg5 protein levels were decreased, leading to autophagy inhibition. Consistently, ATF4 and CREB, two master transcription factors for autophagosome biogenesis, were reduced in TRPM2-depleted cells. In addition, Atg13 and FIP200, which are known to stabilize ULK1 protein, were decreased. Reconstitution with TRPM2 fully restored proliferation, viability, and autophagy; ATF4 and CREB fully restored proliferation and viability but only partially restored autophagy. TRPM2 expression reduced the elevated ROS found in depleted cells. These data show that TRPM2 has an important role in AML proliferation and survival through regulation of key transcription factors and target genes involved in mitochondrial function, bioenergetics, the antioxidant response, and autophagy. Targeting TRPM2 may represent a novel therapeutic approach to inhibit myeloid leukemia growth and enhance susceptibility to chemotherapeutic agents through multiple pathways.

The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress-induced cardiac injury remain unclear. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H2 O2 resulted in 10- to 21-fold increases in Pyk2 phosphorylation in wild-type (WT) myocytes which was significantly lower (~40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (~54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2-mediated Ca2+ increase with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca2+ influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca2+ uptake and enhance adenosine triphosphate generation. Although Trpm2-mediated Ca2+ influx phosphorylated Ca2+ -calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H2 O2 -treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO-I/R when compared with WT-I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria-targeted superoxide scavenger MitoTempo. Our results suggested that Ca2+ influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury.

Cardiac contractility is regulated by the intracellular Ca 2+ concentration fluxes which are actively regulated by multiple channels and transporters. Ca 2+ uptake into the mitochondrial matrix is precisely controlled by the highly Ca 2+ selective channel, Mitochondrial Calcium Uniporter (MCU). Earlier studies on the cardiac-specific acute MCU knockout and a transgenic dominant-negative MCU mice have demonstrated that mitochondrial Ca 2+ ( m Ca 2+ ) signaling is necessary for cardiac ‘‘fight-or-flight’’ contractile response, however, the role of m Ca 2+ buffering to shape global cytosolic Ca 2+ levels and affect E-C coupling, particularly the Ca 2+ transient, on a beat-to-beat basis still remains to be solved. Our earlier studies have demonstrated that loss of MCU Regulator 1 (MCUR1) in cardiomyocytes results in the impaired m Ca 2+ uptake. We have now employed the cardiac-specific MCUR1 knockout mouse to dissect the precise role of MCU in regulating cytosolic Ca 2+ transients associated with excitation-contraction (E-C) coupling and cardiac function. Results from our studies including the in vivo analyses of cardiac physiology during normal and pressure-overloaded mouse models and in vitro experiments including single-cell cardiac contractility, calcium transients, and electrophysiology measurements demonstrate that MCUR1/MCU regulated m Ca 2+ buffering in cardiomyocytes, although insignificant under basal condition, becomes critical in stress induced conditions and actively participates in regulating the c Ca 2+ transients. Also, the ablation of MCUR1 in cardiomyocytes during stress conditions prevents m Ca 2+ overload and subsequent mROS overproduction. Our data indicate that MCUR1 ablation offers protection against pressure-overload cardiac hypertrophy. In summary, our results provide critical insights into the mechanisms by which the MCU channel contributes in regulating the contractile function of the cardiomyocytes and the role of m Ca 2+ in the development and progression of heart failure.

Transient receptor potential melastatin channel subfamily member 2 (TRPM2) has an essential function in cell survival and is highly expressed in many cancers. Inhibition of TRPM2 in neuroblastoma by depletion with CRISPR technology or expression of dominant negative TRPM2-S has been shown to significantly reduce cell viability. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in TRPM2 modulation of neuroblastoma viability was explored. In TRPM2-depleted cells, phosphorylation and expression of Pyk2 and cAMP-responsive element-binding protein (CREB), a downstream target, were significantly reduced after application of the chemotherapeutic agent doxorubicin. Overexpression of wild-type Pyk2 rescued cell viability. Reduction of Pyk2 expression with shRNA decreased cell viability and CREB phosphorylation and expression, demonstrating Pyk2 modulates CREB activation. TRPM2 depletion impaired phosphorylation of Src, an activator of Pyk2, and this may be a mechanism to reduce Pyk2 phosphorylation. TRPM2 inhibition was previously demonstrated to decrease mitochondrial function. Here, CREB, Pyk2, and phosphorylated Src were reduced in mitochondria of TRPM2-depleted cells, consistent with their role in modulating expression and activation of mitochondrial proteins. Phosphorylated Src and phosphorylated and total CREB were reduced in TRPM2-depleted nuclei. Expression and function of mitochondrial calcium uniporter (MCU), a target of phosphorylated Pyk2 and CREB, were significantly reduced. Wild-type TRPM2 but not Ca2+-impermeable mutant E960D reconstituted phosphorylation and expression of Pyk2 and CREB in TRPM2-depleted cells exposed to doxorubicin. Results demonstrate that TRPM2 expression protects the viability of neuroblastoma through Src, Pyk2, CREB, and MCU activation, which play key roles in maintaining mitochondrial function and cellular bioenergetics.

The pathophysiology of human immunodeficiency virus (HIV)-associated cardiomyopathy remains uncertain. We used HIV-1 transgenic (Tg26) mice to explore mechanisms by which HIV-related proteins impacted on myocyte function. Compared to adult ventricular myocytes isolated from nontransgenic (wild type [WT]) littermates, Tg26 myocytes had similar mitochondrial membrane potential (ΔΨ m ) under normoxic conditions but lower Δ Ψ m after hypoxia/reoxygenation (H/R). In addition, Δ Ψ m in Tg26 myocytes failed to recover after Ca 2+ challenge. Functionally, mitochondrial Ca 2+ uptake was severely impaired in Tg26 myocytes. Basal and maximal oxygen consumption rates (OCR) were lower in normoxic Tg26 myocytes, and further reduced after H/R. Complex I subunit and ATP levels were lower in Tg26 hearts. Post-H/R, mitochondrial superoxide (O 2 •- ) levels were higher in Tg26 compared to WT myocytes. Overexpression of B-cell lymphoma 2-associated athanogene 3 (BAG3) reduced O 2 •- levels in hypoxic WT and Tg26 myocytes back to normal. Under normoxic conditions, single myocyte contraction dynamics were similar between WT and Tg26 myocytes. Post-H/R and in the presence of isoproterenol, myocyte contraction amplitudes were lower in Tg26 myocytes. BAG3 overexpression restored Tg26 myocyte contraction amplitudes to those measured in WT myocytes post-H/R. Coimmunoprecipitation experiments demonstrated physical association of BAG3 and the HIV protein Tat. We conclude: (a) Under basal conditions, mitochondrial Ca 2+ uptake, OCR, and ATP levels were lower in Tg26 myocytes; (b) post-H/R, Δ Ψ m was lower, mitochondrial O 2 •- levels were higher, and contraction amplitudes were reduced in Tg26 myocytes; and (c) BAG3 overexpression decreased O 2 •- levels and restored contraction amplitudes to normal in Tg26 myocytes post-H/R in the presence of isoproterenol.

We have previously demonstrated that methylene blue (MB) counteracts the effects of hydrogen sulfide (H(2)S) cardiotoxicity by improving cardiomyocyte contractility and intracellular Ca(2+) homeostasis disrupted by H(2)S poisoning. In vivo, MB restores cardiac contractility severely depressed by sulfide and protects against arrhythmias, ranging from bundle branch block to ventricular tachycardia or fibrillation. To dissect the cellular mechanisms by which MB reduces arrhythmogenesis and improves bioenergetics in myocytes intoxicated with H(2)S, we evaluated the effects of H(2)S on resting membrane potential (E(m)), action potential (AP), Na(+)/Ca(2+) exchange current (I(NaCa)), depolarization-activated K(+) currents and ATP levels in adult mouse cardiac myocytes and determined if MB could counteract the toxic effects of H(2)S on myocyte electrophysiology and ATP. Exposure to toxic concentrations of H(2)S (100 μM) significantly depolarized E(m), reduced AP amplitude, prolonged AP duration at 90% repolarization (APD(90)), suppressed I(NaCa) and depolarization-activated K(+) currents, and reduced ATP levels in adult mouse cardiac myocytes. Treating cardiomyocytes with MB (20 μg/ml) 3 min after H(2)S exposure restored E(m), APD(90), I(NaCa), depolarization-activated K(+) currents, and ATP levels towards normal. MB improved mitochondrial membrane potential (Δψ(m)) and oxygen consumption rate (OCR) in myocytes in which Complex I was blocked by rotenone. We conclude that MB ameliorated H(2)S-induced cardiomyocyte toxicity at multiple levels: (i) reversing excitation-contraction coupling defects (Ca(2+) homeostasis and L-type Ca(2+) channels); (ii) reducing risks of arrhythmias (E(m), APD, I(NaCa) and depolarization-activated K(+) currents); and (iii) improving cellular bioenergetics (ATP, Δψ(m)).

The aim of the present study was to determine whether methylene blue (MB) could directly oppose the neurological toxicity of a lethal cyanide (CN) intoxication. KCN, infused at the rate of 0.375 mg/kg/min intravenously, produced 100% lethality within 15 min in unanaesthetized rats (n = 12). MB at 10 (n = 5) or 20 mg/kg (n = 5), administered 3 min into CN infusion, allowed all animals to survive with no sequelae. No apnea and gasping were observed at 20 mg/kg MB (P < 0.001). The onset of coma was also significantly delayed and recovery from coma was shortened in a dose-dependent manner (median of 359 and 737 seconds, respectively, at 20 and 10 mg/kg). At 4 mg/kg MB (n = 5), all animals presented faster onset of coma and apnea and a longer period of recovery than at the highest doses (median 1344 seconds, P < 0.001). MB reversed NaCN-induced resting membrane potential depolarization and action potential depression in primary cultures of human fetal neurons intoxicated with CN. MB restored calcium homeostasis in the CN-intoxicated human SH-SY5Y neuroblastoma cell line. We conclude that MB mitigates the neuronal toxicity of CN in a dose-dependent manner, preventing the lethal depression of respiratory medullary neurons and fatal outcome.

The mitochondrial calcium uniporter (mtCU) is a ~700 kD multi-subunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. Mitochondrial Calcium Uniporter B ( MCUB ) is reported to negatively regulate m Ca 2+ uptake, but its precise functional role and contribution to cardiac physiology remain unresolved. Size exclusion chromatography of ventricular mitochondria revealed MCUB was absent from high-molecular weight (MW) mtCU complexes in sham animals, but present 24 hours following myocardial ischemia-reperfusion injury (IR). To investigate MCUB ’s contribution to mtCU regulation we created a MCUB -/- cell line by CRISPR-Cas9n. MCUB deletion increased histamine-mediated [ m Ca 2+ ] transient amplitude by ~50% vs. WT controls (mito-R-GECO1). MCUB deletion increased mtCU capacitance (mitoplast patch-clamp) and rate of [ m Ca 2+ ] uptake. Size-exclusion chromatography revealed loss of MCUB increased MCU incorporation into high-MW mtCU, suggesting stoichiometric replacement and overall more functional mtCU’s. To examine MCUB’s role in cardiac physiology we generated a cardiac-specific, tamoxifen-inducible MCUB mouse model (CAG-CAT-MCUB x MCM; MCUB-Tg). FPLC revealed MCUB was undetected in high-MW mtCU complexes of Cre controls, but enriched in MCUB-Tg hearts. MCUB incorporation decreased the presence of channel gatekeepers, MICU1/2, and decreased the MW of the mtCU complex. Immunoprecipitations suggest MCUB interacts with MCU but not MICU1/2. MCUB-Tg adult cardiomyocytes (ACMs) expressing AAV9-mitycam ( m Ca 2+ reporter) were paced and displayed a ~30% decrease in m Ca 2+ transient peak amplitude with significantly reduced m Ca 2+ uptake rates vs controls. A reduction in OxPhos reserve capacity correlated with a severe impairment in cardiac contractile reserve (LV invasive hemodynamics during isoproterenol infusion). MCUB-Tg cardiac mitochondria were resistant to Ca 2+ -induced permeability transition and MCUB-Tg mice displayed ~50% decrease in infarct size per area-at-risk after in vivo IR-injury. These data suggest MCUB regulation of the mtCU is an endogenous compensatory mechanism to decrease m Ca 2+ overload during ischemic injury, but maladaptive to cardiac energetic responsiveness.

In adult mouse myocytes, brief exposure to sodium cyanide (CN) in the presence of glucose does not decrease ATP levels, yet produces profound reduction in contractility, intracellular Ca2+ concentration ([Ca2+]i) transient and L-type Ca2+ current (ICa) amplitudes. We analyzed proteomes from myocytes exposed to CN, focusing on ionic currents associated with excitation-contraction coupling. CN induced phosphorylation of α1c subunit of L-type Ca2+ channel and α2 subunit of Na+-K+-ATPase. Methylene blue (MB), a CN antidote that we previously reported to ameliorate CN-induced reduction in contraction, [Ca2+]i transient and ICa amplitudes, was able to reverse this phosphorylation. CN decreased Na+-K+-ATPase current contributed by α2 but not α1 subunit, an effect that was also counteracted by MB. Peptide consensus sequences suggested CN-induced phosphorylation was mediated by protein kinase C epsilon (PKCε). Indeed, CN stimulated PKC kinase activity and induced PKCε membrane translocation, effects that were prevented by MB. Pretreatment with myristoylated PKCε translocation activator or inhibitor peptides mimicked and inhibited the effects of CN on ICa and myocyte contraction, respectively. We conclude that CN activates PKCε, which phosphorylates L-type Ca2+ channel and Na+-K+-ATPase, resulting in depressed cardiac contractility. We hypothesize that this inhibition of ion fluxes represents a novel mechanism by which the cardiomyocyte reduces its ATP demand (decreased ion fluxes and contractility), diminishes ATP turnover and preserves cell viability. However, this cellular protective effect translates into life-threatening cardiogenic shock in vivo, thereby creating a profound disconnect between survival mechanisms at the cardiomyocyte level from those at the level of the whole organism.

The present study was undertaken to test the hypothesis that gene delivery of BCL2-Associated Athanogene 3 (BAG3) to the heart of mice with left ventricular dysfunction secondary to a myocardial infarction could enhance cardiac performance.BAG3 is a 575 amino acid protein that has pleotropic functions in the cell including pro-autophagy and anti-apoptosis. Mutations in BAG3 have been associated with both skeletal muscle dysfunction and familial dilated cardiomyopathy and BAG3 levels are diminished in non-familial heart failure.Eight-week-old C57/BL6 mice underwent ligation of the left coronary artery (MI) or sham surgery (Sham). Eight weeks later, mice in both groups were randomly assigned to receive either a retro-orbital injection of rAAV9-BAG3 (MI-BAG3 or Sham-BAG3) or rAAV9-GFP (MI-GFP or Sham GFP). Mice were sacrificed at 3 weeks post-injection and myocytes were isolated from the left ventricle.MI-BAG3 mice demonstrated a significantly (p0.0001) higher left ventricular ejection fraction (LVEF) 9 days after rAAV9-BAG3 injection with further improvement in LVEF, fractional shortening and stroke volume at 3 weeks post-injection without changes in LV mass or LV volume. Injection of rAAV9-BAG3 had no effect on LVEF in Sham mice. The salutary benefits of rAAV9-BAG3 were also observed in myocytes isolated from MI hearts including improved cell shortening (p0.05), increased systolic [CaThe results suggest that BAG3 gene therapy may provide a novel therapeutic option for the treatment of heart failure.

Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in modulating cell survival following oxidant injury and is highly expressed in many cancers including neuroblastoma. Here, in xenografts generated from neuroblastoma cells in which TRPM2 was depleted with CRISPR/Cas9 technology and in in vitro experiments, tumor growth was significantly inhibited and doxorubicin sensitivity increased. The hypoxia-inducible transcription factor 1/2α (HIF-1/2α) signaling cascade including proteins involved in oxidant stress, glycolysis, and mitochondrial function was suppressed by TRPM2 depletion. TRPM2-depleted SH-SY5Y neuroblastoma cells demonstrated reduced oxygen consumption and ATP production after doxorubicin, confirming impaired cellular bioenergetics. In cells in which TRPM2 was depleted, mitochondrial superoxide production was significantly increased, particularly following doxorubicin. Ectopic expression of superoxide dismutase 2 (SOD2) reduced ROS and preserved viability of TRPM2-depleted cells, however, failed to restore ATP levels. Mitochondrial reactive oxygen species (ROS) were also significantly increased in cells in which TRPM2 function was inhibited by TRPM2-S, and pretreatment of these cells with the antioxidant MitoTEMPO significantly reduced ROS levels in response to doxorubicin and protected cell viability. Expression of the TRPM2 pore mutant E960D, in which calcium entry through TRPM2 is abolished, also resulted in significantly increased mitochondrial ROS following doxorubicin treatment, showing the critical role of TRPM2-mediated calcium entry. These findings demonstrate the important function of TRPM2 in modulation of cell survival through mitochondrial ROS, and the potential of targeted inhibition of TRPM2 as a therapeutic approach to reduce cellular bioenergetics, tumor growth, and enhance susceptibility to chemotherapeutic agents.

We have previously reported that methylene blue (MB) can counteract hydrogen sulfide (H2S) intoxication-induced circulatory failure. Because of the multifarious effects of high concentrations of H2S on cardiac function, as well as the numerous properties of MB, the nature of this interaction, if any, remains uncertain. The aim of this study was to clarify 1) the effects of MB on H2S-induced cardiac toxicity and 2) whether L-type Ca2+channels, one of the targets of H2S, could transduce some of the counteracting effects of MB. In sedated rats, H2S infused at a rate that would be lethal within 5 min (24 μM·kg−1·min−1), produced a rapid fall in left ventricle ejection fraction, determined by echocardiography, leading to a pulseless electrical activity. Blood concentrations of gaseous H2S reached 7.09 ± 3.53 μM when cardiac contractility started to decrease. Two to three injections of MB (4 mg/kg) transiently restored cardiac contractility, blood pressure, and V̇o2, allowing the animals to stay alive until the end of H2S infusion. MB also delayed PEA by several minutes following H2S-induced coma and shock in unsedated rats. Applying a solution containing lethal levels of H2S (100 μM) on isolated mouse cardiomyocytes significantly reduced cell contractility, intracellular calcium concentration ([Ca2+]i) transient amplitudes, and L-type Ca2+currents ( ICa) within 3 min of exposure. MB (20 mg/l) restored the cardiomyocyte function, ([Ca2+]i) transient, and ICa. The present results offer a new approach for counteracting H2S toxicity and potentially other conditions associated with acute inhibition of L-type Ca2+channels.

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na(+)-K(+)-ATPase and L-type Ca(2+) channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca(2+) channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca(2+)]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca(2+) current (ICa) and sarcoplasmic reticulum (SR) Ca(2+) content but not Na(+)/Ca(2+) exchange current (INaCa) or SR Ca(2+) uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca(2+) entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca(2+) channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure.

Cardiac contractility is regulated by the intracellular Ca 2+ concentration fluxes which are maintained by multiple channels and transporters. Mitochondrial Calcium Uniporter (MCU) is the highly selective Ca 2+ ion channel responsible for the electrophoretic Ca 2+ uptake into the mitochondrial matrix. Earlier studies on the cardiac-specific acute MCU knockout and transgenic dominant-negative MCU mice have demonstrated that mitochondrial Ca 2+ ( m Ca 2+ ) signaling is necessary for cardiac ‘‘fight-or-flight’’ contractile response, however, the role of m Ca 2+ in the beat-to-beat regulation of cardiac contractility is highly debated. The MCU channel function is regulated by varied molecular regulatory components as well as ROS mediated modification at the Cys-96 residue of the core MCU protein. Our earlier studies have demonstrated that loss of MCU Regulator 1 (MCUR1) in cardiomyocytes results in the impaired m Ca 2+ uptake. To dissect the precise role of MCU in regulating cytosolic Ca 2+ transients associated with excitation-contraction (E-C) coupling and cardiac functions, we have employed the loss-of-function, cardiac-specific MCUR1 knockout mouse and a gain-of-function MCU C96A knockin mouse generated by CRISPR/Cas9 approach. Using these novel mouse models, both by in vivo analyses of cardiac physiology and in vitro experiments including single-cell cardiac contractility, calcium transients, and electrophysiology measurements, our study demonstrates that m Ca 2+ buffering actively participates in E-C coupling. MCUR1/MCU regulated m Ca 2+ buffering in cardiomyocytes, although insignificant under basal condition, becomes critical in stress-induced conditions and actively participates in regulating the c Ca 2+ transients during adrenergic stimulation. Our data indicate that MCUR1 ablation offers protection against pressure-overload cardiac hypertrophy and MCU C96A KI mice exhibit significantly decreased LV ejection fraction at baseline. In summary, our results provide critical insights into the mechanisms by which the MCU channel contributes in regulating the contractile function of the cardiomyocytes and the role of m Ca 2+ in the development and progression of heart failure.

The mitochondrial calcium uniporter channel (mtCU) resides in the inner mitochondrial membrane and is a multi-subunit complex required for mitochondrial Ca 2+ ( m Ca 2+ ) uptake. RNA-seq and PCR analysis of mtCU component expression revealed that MCUB expression increases after myocardial infarction. To investigate how MCUB , a paralog of the MCU gene, contributes to mtCU regulation we created a MCUB -/- cell line (HeLa) using CRISPR-Cas9n. MCUB deletion increased histamine-mediated m [Ca 2+ ] transient amplitude by ~50% vs. WT controls (mito-R-GECO). Biophysical characterization found MCUB deletion increased mtCU capacitance and rate of m [Ca 2+ ] uptake. FPLC fractionation of mtCU revealed loss of MCUB increased MCU incorporation into the supercomplex, suggesting MCUB replacement of MCU and overall increase in functional mtCU supercomplexes. Next, we generated a cardiac-specific, tamoxifen-inducible MCUB overexpression mouse model (CAG-CAT-MCUB x MCM; MCUB-Tg) to examine how the MCUB/MCU ratio regulates mtCU function and cardiac physiology. MCUB-Tg mice were infected with AAV9-mitycam (mito genetic calcium reporter) and adult cardiomyocytes were isolated to record [ m Ca 2+ ] transients during pacing. Increasing the MCUB/MCU ratio decreased [ m Ca 2+ ] peak amplitude by ~30% and significantly reduced the [ m Ca 2+ ] uptake rate. FPLC assessment of the mtCU revealed MCUB was undetectable in the supercomplex fraction (~700kD) of Cre controls, but enriched in MCUB -Tg hearts. Examining the oxygen consumption rate of cardiomyocytes isolated from MCUB- Tg hearts found decreased maximal respiration and spare reserve capacity, which correlated with severe impairment in isoproterenol-induced contractile reserve. Mitochondria from MCUB -Tg mouse hearts were resistant to Ca 2+ induced mitochondrial swelling suggesting MCUB protects against permeability transition. MCUB -Tg mice subjected to myocardial ischemia reperfusion injury had a ~50% decrease in infarct size suggesting increased MCUB expression prevents m Ca 2+ overload and limits cell death. These data suggest that MCUB regulation of the mtCU is an endogenous compensatory mechanism to decrease m Ca 2+ overload during ischemic injury, but maladaptive in mitochondrial energetic responsiveness.

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid protein that is found predominantly in the heart, skeletal muscle and many cancers. Deletions and truncations in BAG3 that result in haplo-insufficiency have been associated with the development of dilated cardiomyopathy. To study the cellular and molecular events attributable to BAG3 haplo-insufficiency we generated a mouse in which one allele of BAG3 was flanked by loxP recombination sites (BAG3(fl/+)). Mice were crossed with α-MHC-Cre mice in order to generate mice with cardiac-specific haplo-insufficiency (cBAG3(+/−)) and underwent bi-weekly echocardiography to assess their cardiac phenotype. By ten weeks of age, cBAG3(+/−) mice demonstrated increased heart size and diminished left ventricular ejection fraction when compared with non-transgenic littermates (Cre(−/−)BAG3(fl/+)). Contractility in adult myocytes isolated from cBAG3(+/−) mice were similar to those isolated from control mice at baseline, but showed a significantly decreased response to adrenergic stimulation. Intracellular calcium ([Ca(2+)](i)) transient amplitudes in myocytes isolated from cBAG3(+/−) mice were also similar to myocytes isolated from control mice at baseline but were significantly lower than myocytes from control mice in their response to isoproterenol. BAG3 haplo-insufficiency was also associated with decreased autophagy flux and increased apoptosis. Taken together, these results suggest that mice in which BAG3 has been deleted from a single allele provide a model that mirrors the biology seen in patients with heart failure and BAG3 haplo-insufficiency.

In adult left ventricular mouse myocytes, exposure to sodium cyanide (NaCN) in the presence of glucose dose-dependently reduced contraction amplitude, with ~80% of maximal inhibitory effect attained at 100 µM. NaCN (100 µM) exposure for 10 min significantly decreased contraction and intracellular Ca2+concentration ([Ca2+]i) transient amplitudes, systolic but not diastolic [Ca2+]i, and maximal L-type Ca2+current ( ICa) amplitude, indicating acute alteration of [Ca2+]ihomeostasis largely accounted for the observed excitation-contraction abnormalities. In addition, NaCN depolarized resting membrane potential ( Em), reduced action potential (AP) amplitude, prolonged AP duration at 50% (APD50) and 90% repolarization (APD90), and suppressed depolarization-activated K+currents but had no effect on Na+-Ca2+exchange current ( INaCa). NaCN did not affect cellular adenosine triphosphate levels but depolarized mitochondrial membrane potential (ΔΨm) and increased superoxide (O2·−) levels. Methylene blue (MB; 20 µg/ml) added 3 min after NaCN restored contraction and [Ca2+]itransient amplitudes, systolic [Ca2+]i, Em, AP amplitude, APD50, APD90, ICa, depolarization-activated K+currents, ΔΨm, and O2·−levels toward normal. We conclude that MB reversed NaCN-induced cardiotoxicity by preserving intracellular Ca2+homeostasis and excitation-contraction coupling ( ICa), minimizing risks of arrhythmias ( Em, AP configuration, and depolarization-activated K+currents), and reducing O2·−levels.NEW & NOTEWORTHY Cyanide poisoning due to industrial exposure, smoke inhalation, and bioterrorism manifests as cardiogenic shock and requires rapidly effective antidote. In the early stage of cyanide exposure, adenosine triphosphate levels are normal but myocyte contractility is reduced, largely due to alterations in Ca2+homeostasis because of changes in oxidation-reduction environment of ion channels. Methylene blue, a drug approved by the U.S. Food and Drug Administration, ameliorates cyanide toxicity by normalizing oxidation-reduction state and Ca2+channel function.

Since highly active antiretroviral therapy improved long-term survival of acquired immunodeficiency syndrome (AIDS) patients, AIDS cardiomyopathy has become an increasingly relevant clinical problem. We used human immunodeficiency virus (HIV)-1 transgenic (Tg26) mouse to explore molecular mechanisms of AIDS cardiomyopathy. Tg26 mice had significantly lower left ventricular (LV) mass and smaller end-diastolic and end-systolic LV volumes. Under basal conditions, cardiac contractility and relaxation and single myocyte contraction dynamics were not different between wild-type (WT) and Tg26 mice. Ten days after open heart surgery, contractility and relaxation remained significantly depressed in Tg26 hearts, suggesting that Tg26 mice did not tolerate surgical stress well. To simulate heart failure in which expression of Bcl2-associated athanogene 3 (BAG3) is reduced, we down-regulated BAG3 by small hairpin ribonucleic acid in WT and Tg26 hearts. BAG3 down-regulation significantly reduced contractility in Tg26 hearts. BAG3 overexpression rescued contractile abnormalities in myocytes expressing the HIV-1 protein Tat. We conclude: (i) Tg26 mice exhibit normal contractile function at baseline; (ii) Tg26 mice do not tolerate surgical stress well; (iii) BAG3 down-regulation exacerbated cardiac dysfunction in Tg26 mice; (iv) BAG3 overexpression rescued contractile abnormalities in myocytes expressing HIV-1 protein Tat; and (v) BAG3 may occupy a role in pathogenesis of AIDS cardiomyopathy.

We evaluated whether phospholemman (PLM) regulates L-type Ca2 + current (ICa) in mouse ventricular myocytes. Expression of α1-subunit of L-type Ca2 + channels between wild-type (WT) and PLM knockout (KO) hearts was similar. Compared to WT myocytes, peak ICa (at − 10 mV) from KO myocytes was ~ 41% larger, the inactivation time constant (τinact) of ICa was ~ 39% longer, but deactivation time constant (τdeact) was similar. In the presence of isoproterenol (1 μM), peak ICa was ~ 48% larger and τinact was ~ 144% higher in KO myocytes. With Ba2 + as the permeant ion, PLM enhanced voltage-dependent inactivation but had no effect on τdeact. To dissect the molecular determinants by which PLM regulated ICa, we expressed PLM mutants by adenovirus-mediated gene transfer in cultured KO myocytes. After 24 h in culture, KO myocytes expressing green fluorescent protein (GFP) had significantly larger peak ICa and longer τinact than KO myocytes expressing WT PLM; thereby independently confirming the observations in freshly isolated myocytes. Compared to KO myocytes expressing GFP, KO myocytes expressing the cytoplasmic domain truncation mutant (TM43), the non-phosphorylatable S68A mutant, the phosphomimetic S68E mutant, and the signature PFXYD to alanine (ALL5) mutant all resulted in lower peak ICa. Expressing PLM mutants did not alter expression of α1-subunit of L-type Ca2 + channels in cultured KO myocytes. Our results suggested that both the extracellular PFXYD motif and the transmembrane domain of PLM but not the cytoplasmic tail were necessary for regulation of peak ICa amplitude. We conclude that PLM limits Ca2 + influx in cardiac myocytes by reducing maximal ICa and accelerating voltage-dependent inactivation.

Ubiquitously expressed Trpm2 channel limits oxidative stress and preserves mitochondrial function. We first demonstrated that intracellular Ca2+concentration increase after Trpm2 activation was due to direct Ca2+influx and not indirectly via reverse Na+/Ca2+exchange. To elucidate whether Ca2+entry via Trpm2 is required to maintain cellular bioenergetics, we injected adenovirus expressing green fluorescent protein (GFP), wild-type (WT) Trpm2, and loss-of-function (E960D) Trpm2 mutant into left ventricles of global Trpm2 knockout (gKO) or WT hearts. Five days post-injection, gKO-GFP heart slices had higher reactive oxygen species (ROS) levels but lower oxygen consumption rate (OCR) than WT-GFP heart slices. Trpm2 but not E960D decreased ROS and restored OCR in gKO hearts back to normal levels. In gKO myocytes expressing Trpm2 or its mutants, Trpm2 but not E960D reduced the elevated mitochondrial superoxide (O2.−) levels in gKO myocytes. After hypoxia-reoxygenation (H/R), Trpm2 but not E906D or P1018L (inactivates Trpm2 current) lowered O2.−levels in gKO myocytes and only in the presence of extracellular Ca2+, indicating sustained Ca2+entry is necessary for Trpm2-mediated preservation of mitochondrial function. After ischemic-reperfusion (I/R), cardiac-specific Trpm2 KO hearts exhibited lower maximal first time derivative of LV pressure rise (+dP/d t) than WT hearts in vivo. After doxorubicin treatment, Trpm2 KO mice had worse survival and lower +dP/d t. We conclude 1) cardiac Trpm2-mediated Ca2+influx is necessary to maintain mitochondrial function and protect against H/R injury; 2) Ca2+influx via cardiac Trpm2 confers protection against H/R and I/R injury by reducing mitochondrial oxidants; and 3) Trpm2 confers protection in doxorubicin cardiomyopathy.

Dysregulation of mitochondrial Ca2+-dependent bioenergetics has been implicated in various pathophysiological settings, including neurodegeneration and myocardial infarction. Although mitochondrial Ca2+ transport has been characterized, and several molecules, including LETM1, have been identified, the functional role of LETM1-mediated Ca2+ transport remains unresolved. This study examines LETM1-mediated mitochondrial Ca2+ transport and bioenergetics in multiple cell types, including fibroblasts derived from patients with Wolf-Hirschhorn syndrome (WHS). The results show that both mitochondrial Ca2+ influx and efflux rates are impaired in LETM1 knockdown, and similar phenotypes were observed in ΔEF hand, D676A D688KLETM1 mutant-overexpressed cells, and in cells derived from patients with WHS. Although LETM1 levels were lower in WHS-derived fibroblasts, the mitochondrial Ca2+ uniporter components MCU, MCUR1, and MICU1 remain unaltered. In addition, the MCU mitoplast patch-clamp current (IMCU) was largely unaffected in LETM1-knockdown cells. Silencing of LETM1 also impaired basal mitochondrial oxygen consumption, possibly via complex IV inactivation and ATP production. Remarkably, LETM1 knockdown also resulted in increased reactive oxygen species production. Further, LETM1 silencing promoted AMPK activation, autophagy, and cell cycle arrest. Reconstitution of LETM1 or antioxidant overexpression rescued mitochondrial Ca2+ transport and bioenergetics. These findings reveal the role of LETM1-dependent mitochondrial Ca2+ flux in shaping cellular bioenergetics.—Doonan, P J., Chandramoorthy, H. C., Hoffman, N. E., Zhang, X., Cárdenas, C., Shanmughapriya, S., Rajan, S., Vallem, S., Chen, X., Foskett, J. K., Cheung, J. Y., Houser, S. R., Madesh, M. LETM1-dependent mitochondrial Ca2+ flux modulates cellular bioenergetics and proliferation.

Phospholemman (PLM), when phosphorylated at Ser68, inhibits cardiac Na+/Ca2+exchanger 1 (NCX1) and relieves its inhibition on Na+-K+-ATPase. We have engineered mice in which expression of the phosphomimetic PLM S68E mutant was induced when dietary doxycycline was removed at 5 wk. At 8–10 wk, compared with noninduced or wild-type hearts, S68E expression in induced hearts was ∼35–75% that of endogenous PLM, but protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase, α1- and α2-subunits of Na+-K+-ATPase, α1c-subunit of L-type Ca2+channel, and phosphorylated ryanodine receptor were unchanged. The NCX1 protein level was increased by ∼47% but the NCX1 current was depressed by ∼34% in induced hearts. Isoproterenol had no effect on NCX1 currents but stimulated Na+-K+-ATPase currents equally in induced and noninduced myocytes. At baseline, systolic intracellular Ca2+concentrations ([Ca2+]i), sarcoplasmic reticulum Ca2+contents, and [Ca2+]itransient and contraction amplitudes were similar between induced and noninduced myocytes. Isoproterenol stimulation resulted in much higher systolic [Ca2+]i, sarcoplasmic reticulum Ca2+content, and [Ca2+]itransient and contraction amplitudes in induced myocytes. Echocardiography and in vivo close-chest catheterization demonstrated similar baseline myocardial function, but isoproterenol induced a significantly higher +dP/d t in induced compared with noninduced hearts. In contrast to the 50% mortality observed in mice constitutively overexpressing the S68E mutant, induced mice had similar survival as wild-type and noninduced mice. After ischemia-reperfusion, despite similar areas at risk and left ventricular infarct sizes, induced mice had significantly higher +dP/d t and −dP/d t and lower perioperative mortality compared with noninduced mice. We propose that phosphorylated PLM may be a novel therapeutic target in ischemic heart disease.

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.

Summary: Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (mCa2+) uptake, delays cytosolic Ca2+ (cCa2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid mCa2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism. : Hepatic mitochondrial Ca2+ shapes bioenergetics and lipid homeostasis. Tomar et al. demonstrate that MCU-mediated cCa2+ buffering serves as a crucial step in controlling hepatic fuel metabolism through an MCU/PP4/AMPK molecular cascade. Identification of these molecular signaling events aids in understanding how perturbation of mitochondrial ion homeostasis may contribute to the etiology of metabolic disorders. Keywords: mitochondrial Ca2+ uniporter, calcium, bioenergetics, AMPK, MCU, hepatocyte, lipid metabolism, phosphatase, metabolic diseases, diabetes

tHigh-power impulse magnetron sputtering (HiPIMS), coupled with a direct-current magnetron sputter-ing (dcMS) in parallel, was employed to fabricate graphite-like amorphous carbon (GLC) films. Differentimpulse voltages were applied in HiPIMS during the film deposition. The structure and mechanical prop-erties of the GLC films deposited by the HiPIMS were investigated. The bonding structure of the films wasanalyzed by X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy. Atomic force microscopy(AFM) and Nano-indentation were used to characterize the surface quality and micro-hardness, respec-tively. Internal stress of the films was calculated based on the curvature measured by a laser tester.Tribological behavior of the GLC films is studied by a ball-on-disk tribometer in ambient condition. Theeffects of impulse voltage on deposition rate, internal stress, mechanical and tribological properties ofthe GLC films were investigated. The results are analyzed and discussed.

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca2+ influx that was dependent on extracellular Ca2+, was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/d t between WT and KO hearts. At 2–3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/d t compared with WT hearts. Compared with WT I/R myocytes, expression of the Na+/Ca2+ exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na+-K+-ATPase and Na+ pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca2+ concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

Mitochondrial Ca(2+) Uniporter (MCU)-dependent mitochondrial Ca(2+) uptake is the primary mechanism for increasing matrix Ca(2+) in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here, we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1 (MCUR1) have severely impaired [Ca(2+)]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation, and migration but elicited autophagy. These studies establish the existence of a MCU complex that assembles at the mitochondrial integral membrane and regulates Ca(2+)-dependent mitochondrial metabolism.

The Cr-doped hydrogen-GLC films were prepared by a hybrid magnetron sputtering system composed of a direct current magnetron sputtering (dc-MS) source of carbon and a high power impulse magnetron sputtering, (HIPIMS) source of Cr with reactive gas of C2H2. The hydrogen-free GLC and Cr-doped GLC films were also prepared for comparison. In this paper, the influence of substrate bias on the Cr-doped hydrogen-GLC films was investigated. With the increase of substrate bias from -100 V to -250 V, the re-sputtering of weak bonding sp2 firstly occurred and induced an increased sp3 bonding; however, the following sp3 to sp2 transformation result in a decreased sp3 bonding. The change trend of surface roughness and friction coefficient with the increased bias voltages were the same as that of sp3 bond. The lowest surface roughness and lowest friction coefficient corresponded to the highest sp3 with the Cr-GLC-H films at the bias voltage of -100 V.

Phospholemman (PLM) regulates [Na+]i, [Ca2+]i and contractility through its interactions with Na+‐K+‐ATPase (NKA) and Na+/Ca2+ exchanger (NCX1) in the heart. Both expression and phosphorylation of PLM are altered after myocardial infarction (MI) and heart failure. We tested the hypothesis that absence of PLM regulation of NKA and NCX1 in PLM‐knockout (KO) mice is detrimental. Three weeks after MI, wild‐type (WT) and PLM‐KO hearts were similarly hypertrophied. PLM expression was lower but fractional phosphorylation was higher in WT‐MI compared to WT‐sham hearts. Left ventricular ejection fraction was severely depressed in WT‐MI but significantly less depressed in PLM‐KO‐MI hearts despite similar infarct sizes. Compared with WT‐sham myocytes, the abnormal [Ca2+], transient and contraction amplitudes observed in WT‐MI myocytes were ameliorated by genetic absence of PLM. In addition, NCX1 current was depressed in WT‐MI but not in PLM‐KO‐MI myocytes. Despite improved myocardial and myocyte performance, PLM‐KO mice demonstrated reduced survival after MI. Our findings indicate that alterations in PLM expression and phosphorylation are important adaptations post‐MI, and that complete absence of PLM regulation of NKA and NCX1 is detrimental in post‐MI animals. Clin Trans Sci 2012; Volume #: 1–8

Expression and activity of cardiac Na+/Ca2+ exchanger (NCX1) are altered in many disease states. We engineered mice in which the phosphomimetic phospholemman S68E mutant (inhibits NCX1 but not Na+-K+-ATPase) was constitutively overexpressed in a cardiac-specific manner (conS68E). At 4–6 wk, conS68E mice exhibited severe bradycardia, ventricular arrhythmias, increased left ventricular (LV) mass, decreased cardiac output (CO), and ∼50% mortality compared with wild-type (WT) littermates. Protein levels of NCX1, calsequestrin, ryanodine receptor, and α1- and α2-subunits of Na+-K+-ATPase were similar, but sarco(endo)plasmic reticulum Ca2+-ATPase was lower, whereas L-type Ca2+ channels were higher in conS68E hearts. Resting membrane potential and action potential amplitude were similar, but action potential duration was dramatically prolonged in conS68E myocytes. Diastolic intracellular Ca2+ ([Ca2+]i) was higher, [Ca2+]i transient and maximal contraction amplitudes were lower, and half-time of [Ca2+]i transient decline was longer in conS68E myocytes. Intracellular Na+ reached maximum within 3 min after isoproterenol addition, followed by decline in WT but not in conS68E myocytes. Na+/Ca2+ exchange, L-type Ca2+, Na+-K+-ATPase, and depolarization-activated K+ currents were decreased in conS68E myocytes. At 22 wk, bradycardia and increased LV mass persisted in conS68E survivors. Despite comparable baseline CO, conS68E survivors at 22 wk exhibited decreased chronotropic, inotropic, and lusitropic responses to isoproterenol. We conclude that constitutive overexpression of S68E mutant was detrimental, both in terms of depressed cardiac function and increased arrhythmogenesis.

Using split cardiac Na+/Ca2+exchangers (NCX1), we previously demonstrated that phospholemman (PLM) regulates NCX1 by interacting with the proximal linker domain (residues 218–358) of the intracellular loop of NCX1. With the use of overlapping loop deletion mutants, interaction sites are localized to two regions spanning residues 238–270 and residues 300–328 of NCX1. In this study, we used alanine (Ala) linker scanning to pinpoint the residues in the proximal linker domain involved in regulation of NCX1 by PLM. Transfection of human embryonic kidney (HEK)293 cells with wild-type (WT) NCX1 or its Ala mutants but not empty vector resulted in NCX1 current ( INaCa). Coexpression of PLM with WT NCX1 inhibited INaCa. Mutating residues 248–252 (PASKT) or 300–304 (QKHPD) in WT NCX1 to Ala resulted in loss of inhibition of INaCaby PLM. By contrast, inhibition of INaCaby PLM was preserved when residues 238–242, 243–247, 253–257, 258–262, 263–267, 305–309, 310–314, 315–319, 320–324, or 325–329 were mutated to Ala. While mutating residue 301 to alanine completely abolished PLM inhibition, mutation of any single residue 250–252, 300, or 302–304 resulted in partial reduction in inhibition. Mutating residues 248–252 to Ala resulted in significantly weaker association with PLM. The NCX1-G503P mutant that lacks Ca2+-dependent activation retained its sensitivity to PLM. We conclude that residues 248–252 and 300–304 in the proximal linker domain of NCX1 were involved in its inhibition by PLM.

Ornithine decarboxylase (ODC), the first enzyme of polyamine metabolism, is rapidly upregulated in response to agents that induce a pathological cardiac hypertrophy. Transgenic mice overexpressing ODC in the heart (MHC-ODC mice) experience a much more dramatic left ventricular hypertrophy in response to β-adrenergic stimulation with isoproterenol (ISO) compared to wild-type (WT) controls. ISO also induced arginase activity in transgenic hearts but not in controls. The current work studies the cooperation between the cardiac polyamines and L-arginine (L-Arg) availability in MHC-ODC mice. Although ISO-induced hypertrophy is well-compensated, MHC-ODC mice administered L-Arg along with ISO showed a rapid onset of systolic dysfunction and died within 48 h. Myocytes isolated from MHC-ODC mice administered L-Arg/ISO exhibited reduced contractility and altered calcium transients, suggesting an alteration in [Ca(2+)] homeostasis, and abbreviated action potential duration, which may contribute to arrhythmogenesis. The already elevated levels of spermidine and spermine were not further altered in MHC-ODC hearts by L-Arg/ISO treatment, suggesting alternative L-Arg utilization pathways lead to dysregulation of intracellular calcium. MHC-ODC mice administered an arginase inhibitor (Nor-NOHA) along with ISO died almost as rapidly as L-Arg/ISO-treated mice, while the iNOS inhibitor S-methyl-isothiourea (SMT) was strongly protective against L-Arg/ISO. These results point to the induction of arginase as a protective response to β-adrenergic stimulation in the setting of high polyamines. Further, NO generated by exogenously supplied L-Arg may contribute to the lethal consequences of L-Arg/ISO treatment. Since considerable variations in human cardiac polyamine and L-Arg content are likely, it is possible that alterations in these factors may influence myocyte contractility.

Phospholemman (PLM), a member of the FXYD family of regulators of ion transport, is a major sarcolemmal substrate for protein kinases A and C in cardiac and skeletal muscle. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. Functionally, when phosphorylated at serine(68), PLM stimulates Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger in cardiac myocytes. In heterologous expression systems, PLM modulates the gating of cardiac L-type Ca(2+) channel. Therefore, PLM occupies a key modulatory role in intracellular Na(+) and Ca(2+) homeostasis and is intimately involved in regulation of excitation-contraction (EC) coupling. Genetic ablation of PLM results in a slight increase in baseline cardiac contractility and prolongation of action potential duration. When hearts are subjected to catecholamine stress, PLM minimizes the risks of arrhythmogenesis by reducing Na(+) overload and simultaneously preserves inotropy by inhibiting Na(+)/Ca(2+) exchanger. In heart failure, both expression and phosphorylation state of PLM are altered and may partly account for abnormalities in EC coupling. The unique role of PLM in regulation of Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and potentially L-type Ca(2+) channel in the heart, together with the changes in its expression and phosphorylation in heart failure, make PLM a rational and novel target for development of drugs in our armamentarium against heart failure. Clin Trans Sci 2010; Volume 3: 189-196.

Transient Receptor Potential Melastatin subfamily member 2 (TRPM2) is highly expressed in many cancers including neuroblastoma. Proline-rich tyrosine kinase 2 (Pyk2) is calcium-sensitive non-receptor tyrosine kinase which enhances cell survival and is activated by calcium influx through TRPM2. Pyk2 also modulates mitochondrial calcium uptake through phosphorylation of the mitochondrial calcium uniporter (MCU), impacting mitochondrial function, and through its effects on CREB, which regulates transcription of MCU. The purpose of this work was to investigate the mechanisms by which TRPM2 regulates cancer cell survival in order to develop new therapeutic strategies based on inhibition of TRPM2. Two models of TRPM2 inhibition were utilized: 1) reducing TRPM2 function in SH-SY5Y cells by expression of the dominant negative construct TRPM2-S, a short isoform of TRPM2, and 2) depletion of TRPM2 expression with CRISPR/Cas9 technology. Treatment with Doxorubicin (Doxo) was used as a model of oxidative stress. Doxorubicin reduced growth of cells in which TRPM2 was inhibited significantly more than control cells. Inhibition of TRPM2 also reduced Pyk2 and CREB expression and activation. Using shRNA technology we showed that Pyk2 mediated reduced expression and activation of CREB and activation of STAT3 in TRPM2 expressing cells. Fractionation experiments showed that depletion of TRPM2 reduced phosphorylation of whole cell and mitochondrial Src and Pyk2 and expression of mitochondrial CREB and MCU. Src has an important role in Pyk2 phosphorylation. In TRPM2 depleted cells, mitochondrial Ca uptake was significantly reduced since both peak mitochondrial calcium uniporter currents and amount of calcium transported (current-time integral) measured in mitoplasts were lower. Wt Pyk2 but not the phospho-deficient mutant Y402F and wt TRPM2 but not the TRPM2 Ca-impermeant mutant E960D, restored viability after Doxo treatment of SH-SY5Y cells in which TRPM2 was inhibited. In conclusion, these results suggest that TRPM2 inhibition leads to reduced viability in neuroblastoma through the Src-Pyk2-CREB-MCU pathway, which plays a key role in regulating mitochondrial function and cellular bioenergetics. Citation Format: Iwona Malgorzata Hirschler-Laszkiewicz, Shu-jen Chen, Lei Bao, JuFang Wang, Xue-Qian Zhang, Santhanam Shanmughapriya, Kerry Keefer, Muniswamy Madesh, Joseph Y. Cheung, Barbara A. Miller. TRPM2 modulates neuroblastoma cell survival through Pyk2, CREB and MCU activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1458.

Acute myeloid leukemia (AML) is a heterogeneous group of leukemias that arise in hematopoietic precursors. Understanding the mechanisms involved in AML proliferation and survival is key to development of new therapies. Transient receptor potential melastatin 2 (TRPM2), a calcium permeable ion channel, modulates cell proliferation, survival and oxidative stress-induced cell death and is highly expressed in many cancer cells including AML. To investigate the role of TRPM2 in AML, we depleted the TRPM2 gene in the human AML cell line U937 using clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR) technology. RT-PCR, western blot, and patch clamp analyses confirmed that TRPM2 was depleted in U937 knockout (KO) cells. Cell proliferation and viability measured by XTT and trypan blue exclusion assays were significantly reduced in KO cells after doxorubicin treatment. Expression of wild type TRPM2 (TRPM2-L) restored cell growth and cell survival to doxorubicin-treated TRPM2 KO cells, whereas the TRPM2 channel pore mutant (E960D) or empty vector did not. In a xenograft mouse model, mice were injected with TRPM2 KO or control U937 cells which express luciferase. Mice injected with TRPM2 KO U937 cells showed significantly reduced leukemia growth and enhanced sensitivity to doxorubicin. To examine the mechanisms, mitochondrial ROS were measured with confocal microscopy and MitoSOX Red. ROS were significantly elevated in TRPM2 KO cells compared to scrambled control, and further increased when KO cells were treated with doxorubicin. ATP production, oxygen consumption rate (OCR), and mitochondrial Ca2+ uptake were significantly reduced and mitochondrial membrane potential (ΔΨ) significantly increased in TRPM2 KO cells compared to scrambled control cells treated with doxorubicin, demonstrating dysfunctional mitochondria. Western blot analysis showed that expressions of HIF1/2α, FOXO3a and Nrf2 were downregulated in TRPM2 KO cells, further contributing to the increase in ROS. Expression of autophagy proteins ULK1, Atg7 and Atg5 was significantly reduced and mitochondrial protein Tom20 significantly increased in TRPM2 KO cells, suggesting that depletion of TRPM2 significantly reduced autophagy in AML cells. Reconstitution of wild type TRPM2 but not E960D restored ULK1 expression. In conclusion, depletion of TRPM2 significantly inhibits AML proliferation and increases sensitivity to doxorubicin by reducing mitochondrial function, increasing ROS, and impairing autophagy. TRPM2 contributes significantly to AML survival and may be a novel therapeutic target in leukemia therapy. Citation Format: Shu-Jen Chen, Lei Bao, Kerry Keefer, Longgui Chen, JuFang Wang, Xue-Qian Zhang, Santhanam Shanmughapriya, Iwona Hirschler-Laszkiewicz, Muniswamy Madesh, Joseph Y. Cheung, Hong-Gang Wang, Barbara A. Miller. TRPM2 promotes leukemia proliferation and survival through modulation of ROS and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1324.

We have produced mice in which expression of the rat cardiac Na+/Ca2+ exchanger (NCX1) transgene was switched on when doxycycline was removed from the feed at 5 wk. At 8 to 10 wk, NCX1 expression in induced (Ind) mouse hearts was 2.5-fold higher but protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase, α1- and α2-subunits of Na+-K+-ATPase, phospholamban, ryanodine receptor, calsequestrin, and unphosphorylated and phosphorylated phospholemman were unchanged compared with wild-type (WT) or noninduced (non-Ind) hearts. There was no cellular hypertrophy since WT, non-Ind, and Ind myocytes had similar whole cell membrane capacitance. In Ind myocytes, NCX1 current amplitude was ∼42% higher, L-type Ca2+ current amplitude was unchanged, and action potential duration was prolonged compared with WT or non-Ind myocytes. Contraction and intracellular Ca2+ concentration ([Ca2+]i) transient amplitudes in Ind myocytes were lower at 0.6, not different at 1.8, and higher at 5.0 mM extracellular Ca2+ concentration ([Ca2+]o) compared with WT or non-Ind myocytes. Despite similar Ca2+ current amplitude and sarcoplasmic reticulum (SR) Ca2+ uptake, SR Ca2+ content at 5.0 mM [Ca2+]o was significantly higher in Ind compared with non-Ind myocytes, indicating that NCX1 directly contributed to SR Ca2+ loading. Echocardiography demonstrated that heart rate, left ventricular mass, ejection fraction, stroke volume, and cardiac output were similar among the three groups of animals. In vivo close-chest catheterization demonstrated similar contractility and relaxation among the three groups of mice, both at baseline and after stimulation with isoproterenol. We conclude that induced expression of NCX1 transgene resulted in altered [Ca2+]i homeostasis, myocyte contractility, and action potential morphology. In addition, heart failure did not occur 3 to 5 wk after NCX1 transgene was induced to be expressed at levels found in diseased hearts.

Phospholemman (PLM) belongs to the FXYD family of small ion transport regulators. When phosphorylated at Ser68, PLM inhibits cardiac Na+/Ca2+exchanger (NCX1). We previously demonstrated that the cytoplasmic tail of PLM interacts with the proximal intracellular loop (residues 218–358), but not the transmembrane (residues 1–217 and 765–938) or Ca2+-binding (residues 371–508) domains, of NCX1. In this study, we used intact Na+/Ca2+exchanger with various deletions in the intracellular loop to map the interaction sites with PLM. We first demonstrated by Western blotting and confocal immunofluorescence microscopy that wild-type (WT) NCX1 and its deletion mutants were expressed in transfected HEK-293 cells. Cotransfection with PLM and NCX1 (or its deletion mutants) in HEK-293 cells did not decrease expression of NCX1 (or its deletion mutants). Coexpression of PLM with WT NCX1 inhibited NCX1 current ( INaCa). Deletion of residues 240–679, 265–373, 250–300, or 300–373 from WT NCX1 resulted in loss of inhibition of INaCaby PLM. Inhibition of INaCaby PLM was preserved when residues 229–237, 270–300, 328–330, or 330–373 were deleted from the intracellular loop of NCX1. These results suggest that PLM mediated inhibition of INaCaby interacting with two distinct regions (residues 238–270 and 300–328) of NCX1. Indeed, INaCameasured in mutants lacking residues 238–270, 300–328, or 238–270 + 300–328 was not affected by PLM. Glutathione S-transferase pull-down assays confirmed that PLM bound to fragments corresponding to residues 218–371, 218–320, 218–270, 238–371, and 300–373, but not to fragments encompassing residues 250–300 and 371–508 of NCX1, indicating that residues 218–270 and 300–373 physically associated with PLM. Finally, acute regulation of INaCaby PLM phosphorylation observed with WT NCX1 was absent in 250–300 deletion mutant but preserved in 229–237 deletion mutant. We conclude that PLM mediates its inhibition of NCX1 by interacting with residues 238–270 and 300–328.

Phospholemman (PLM) regulates cardiac Na+/Ca2+ exchanger (NCX1) and Na+-K+-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser68, disinhibits Na+-K+-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na+-K+-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca2+-ATPase levels, and retained near normal contractility, but α1-subunit of Na+-K+-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca2+ concentration ([Ca2+]i) transients reverted back to those observed in cultured WT myocytes. Both Na+-K+-ATPase current ( Ipump) and Na+/Ca2+ exchange current ( INaCa) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of INaCa but had no effect on Ipump. Contractility, [Ca2+]i transient amplitudes, and sarcoplasmic reticulum Ca2+ contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on INaCa but decreased Ipump. Contractility, [Ca2+]i transient amplitudes, and sarcoplasmic reticulum Ca2+ contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca2+]i homeostasis primarily by its direct inhibitory effects on Na+/Ca2+ exchange.

Summary Cardiac contractility is mediated by a variable flux in intracellular calcium (Ca 2+ ), thought to be integrated into mitochondria via the mitochondrial calcium uniporter (MCU) channel to match energetic demand. Here, we examine a conditional, cardiomyocyte-specific, mutant mouse lacking Mcu , the pore-forming subunit of the MCU channel, in adulthood. Mcu −/− mice display no overt baseline phenotype and are protected against m Ca 2+ overload in an in vivo myocardial ischemia-reperfusion injury model by preventing the activation of the mitochondrial permeability transition pore, decreasing infarct size, and preserving cardiac function. In addition, we find that Mcu −/− mice lack contractile responsiveness to acute β-adrenergic receptor stimulation and in parallel are unable to activate mitochondrial dehydrogenases and display reduced bioenergetic reserve capacity. These results support the hypothesis that MCU may be dispensable for homeostatic cardiac function but required to modulate Ca 2+ -dependent metabolism during acute stress.

Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+ -permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+ -regulated transcription factor CREB (cyclic adenosine monophosphate response element-binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+ -dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.

Ubiquitously expressed Trpm2 channel limits oxidative stress and preserves mitochondrial function. We first demonstrated that intracellular Ca(2+) concentration increase after Trpm2 activation was due to direct Ca(2+) influx and not indirectly via reverse Na(+)/Ca(2+) exchange. To elucidate whether Ca(2+) entry via Trpm2 is required to maintain cellular bioenergetics, we injected adenovirus expressing green fluorescent protein (GFP), wild-type (WT) Trpm2, and loss-of-function (E960D) Trpm2 mutant into left ventricles of global Trpm2 knockout (gKO) or WT hearts. Five days post-injection, gKO-GFP heart slices had higher reactive oxygen species (ROS) levels but lower oxygen consumption rate (OCR) than WT-GFP heart slices. Trpm2 but not E960D decreased ROS and restored OCR in gKO hearts back to normal levels. In gKO myocytes expressing Trpm2 or its mutants, Trpm2 but not E960D reduced the elevated mitochondrial superoxide (O2(.-)) levels in gKO myocytes. After hypoxia-reoxygenation (H/R), Trpm2 but not E906D or P1018L (inactivates Trpm2 current) lowered O2(.-) levels in gKO myocytes and only in the presence of extracellular Ca(2+), indicating sustained Ca(2+) entry is necessary for Trpm2-mediated preservation of mitochondrial function. After ischemic-reperfusion (I/R), cardiac-specific Trpm2 KO hearts exhibited lower maximal first time derivative of LV pressure rise (+dP/dt) than WT hearts in vivo. After doxorubicin treatment, Trpm2 KO mice had worse survival and lower +dP/dt. We conclude 1) cardiac Trpm2-mediated Ca(2+) influx is necessary to maintain mitochondrial function and protect against H/R injury; 2) Ca(2+) influx via cardiac Trpm2 confers protection against H/R and I/R injury by reducing mitochondrial oxidants; and 3) Trpm2 confers protection in doxorubicin cardiomyopathy.