ABSTRACTBackgroundMitochondrial calcium (mCa2+) uptake through the mitochondrial calcium uniporter channel (mtCU) stimulates metabolism to meet acute increases in cardiac energy demand. However, excessivemCa2+uptake during stress, as in ischemia-reperfusion, initiates permeability transition and cell death. Despite these often-reported acute physiological and pathological effects, a major unresolved controversy is whether mtCU-dependentmCa2+uptake and long-term elevation of cardiomyocytemCa2+contributes to the heart’s adaptation during sustained increases in workload.ObjectiveWe tested the hypothesis that mtCU-dependentmCa2+uptake contributes to cardiac adaptation and ventricular remodeling during sustained catecholaminergic stress.MethodsMice with tamoxifen-inducible, cardiomyocyte-specific gain (αMHC-MCM x flox-stop-MCU; MCU-Tg) or loss (αMHC-MCM xMcufl/fl;Mcu-cKO) of mtCU function received 2-wk catecholamine infusion.ResultsCardiac contractility increased after 2d of isoproterenol in control, but notMcu-cKO mice. Contractility declined and cardiac hypertrophy increased after 1-2-wk of isoproterenol in MCU-Tg mice. MCU-Tg cardiomyocytes displayed increased sensitivity to Ca2+- and isoproterenol-induced necrosis. However, loss of the mitochondrial permeability transition pore (mPTP) regulator cyclophilin D failed to attenuate contractile dysfunction and hypertrophic remodeling, and increased isoproterenol-induced cardiomyocyte death in MCU-Tg mice.ConclusionsmtCUmCa2+uptake is required for early contractile responses to adrenergic signaling, even those occurring over several days. Under sustained adrenergic load excessive MCU-dependentmCa2+uptake drives cardiomyocyte dropout, perhaps independent of classical mitochondrial permeability transition pore opening, and compromises contractile function. These findings suggest divergent consequences for acute versus sustainedmCa2+loading, and support distinct functional roles for the mPTP in settings of acutemCa2+overload versus persistentmCa2+stress.

Heart failure (HF) is a leading cause of hospitalization and mortality worldwide. Yet, there is still limited knowledge on the underlying molecular mechanisms, because human tissue for research is scarce, and data obtained in animal models is not directly applicable to humans. Thus, studies of human heart specimen are of particular relevance. Mitochondrial Ca2+ handling is an emerging topic in HF progression because its regulation is central to the energy supply of the heart contractions as well as to avoiding mitochondrial Ca2+ overload and the ensuing cell death induction. Notably, animal studies have already linked impaired mitochondrial Ca2+ transport to the initiation/progression of HF. Mitochondrial Ca2+ uptake is mediated by the Ca(2+)uniporter (mtCU) that consists of the MCU pore under tight control by the Ca2+-sensing MICU1 and MICU2. The MICU1/ MCU protein ratio has been validated as a predictor of the mitochondrial Ca(2+)uptake phenotype. We here determined for the first time the protein composition of the mtCU in the human heart. The two regulators MICU1 and MICU2, were elevated in the failing human heart versus non-failing controls, while the MCU density was unchanged. Furthermore, the MICU1/MCU ratio was significantly elevated in the failing human hearts, suggesting altered gating of the MCU by MICU1 and MICU2. Based on a small cohort of patients, the decrease in the cardiac contractile function (ejection fraction) seems to correlate with the increase in MICU1/ MCU ratio. Our findings therefore indicate a possible role for adaptive/maladaptive changes in the mtCU composition in the initiation/progression of human HF in humans and point to a potential therapeutic target at the level of the MICU1-dependent regulation of the mtCU.

Rapid mitochondrial calcium (mCa2+) uptake through the mitochondrial calcium uniporter channel (mtCU) matches ATP production to acute increases in cardiac workload and energy demand, but excessive Ca2+ uptake during stress such as ischemia-reperfusion injury initiates permeability transition and cell death.

Pharmacologic activation of branched chain amino acid (BCAA) catabolism is protective in numerous models of heart failure (HF). How this protection occurs has remained unclear, although a causative block in cardiac BCAA oxidation has been proposed. We use here in vivo heavy isotope infusion studies to show that cardiac preference for BCAA oxidation increases, rather than decreases, in multiple models of HF. We use various genetic models to show that cardiac-specific activation of BCAA oxidation does not protect from HF, even though systemic activation of BCAA oxidation does. Lowering plasma and cardiac BCAAs by genetic means is also not sufficient to confer protection comparable to that conferred by pharmacologic activation of BCAA oxidation, suggesting alternative mechanisms of protection. Surprisingly, telemetry and invasive hemodynamic studies showed that pharmacological activation of BCAA catabolism lowers blood pressure, a well-established cardioprotective mechanism. The effects on blood pressure occurred independently of nitric oxide (NO), and reflected a vascular resistance to adrenergic constriction. Finally, mendelian randomization studies revealed that elevations in plasma BCAAs portend higher blood pressure in large human cohorts. Together, these data indicate that activation of BCAA oxidation lowers blood pressure and protects from heart failure independently of any direct effects on the heart itself.

Acute mitochondrial calcium ( m Ca 2+ ) uptake stimulates bioenergetics to meet increased ATP demand, but when excessive predisposes to necrotic cell death. A major unresolved controversy is whether chronic alterations in cardiomyocyte m Ca 2+ homeostasis contribute to maladaptive remodeling and contractile dysfunction in non-ischemic heart disease. We hypothesized that cardiomyocyte m Ca 2+ accumulation drives cardiac maladaptation in response to stressors that chronically increase workload and cytosolic Ca 2+ cycling. We subjected mice with adult, cardiomyocyte-specific manipulation of m Ca 2+ uptake through the mitochondrial calcium uniporter ( Mcu deletion, Mcu -cKO; MCU overexpression, MCU-Tg) or m Ca 2+ efflux through the mitochondrial sodium-calcium exchanger, NCLX (NCLX overexpression, NCLX-OE), to chronic pressure or neurohormonal overload. Fractional shortening failed to increase in Mcu -cKO mice over the first days of isoproterenol (Iso) infusion. Mortality was increased in Mcu -cKO mice over this period, and this effect was recapitulated in NCLX-OE mice infused with angiotensin II + phenylephrine (PE), although contractility did not decline in either case. Hypertrophic responses to chronic stress were attenuated in NCLX-OE but not Mcu -cKO hearts, and adenoviral NCLX expression limited mitochondrial metabolism, protein synthesis, and cell growth in neonatal rat cardiomyocytes treated with PE. These data indicate that m Ca 2+ accumulation is required for cardiac hypertrophy, but MCU is not. MCU-Tg hearts decompensated towards failure with 1-2 weeks of Iso. Although these hearts exhibited increased cardiomyocyte necrosis, deletion of the mPTP regulator cyclophilin D failed to rescue contractility, suggesting that m Ca 2+ overload causes cardiac failure, even independent of permeability transition. Fitting with this view, NCLX-OE attenuated the decline in contractile function that occurred with 12-week pressure overload. We conclude that despite initial adaptive effects, sustained m Ca 2+ elevation drives the progression of non-ischemic heart disease triggered by a chronic increase in cardiac workload. Our findings raise concern over proposed therapeutic strategies aiming to augment m Ca 2+ accumulation in heart failure.

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

The mitochondrial calcium uniporter (MCU) forms the pore of the mitochondrial calcium uniporter channel (mtCU) and is required for rapid mitochondrial Ca 2+ ( m Ca 2+ ) uptake. MCU is necessary to increase cardiac energetics to fuel an increase in cardiac contractility during acute sympathetic stimulation. However, little is known about how MCU-dependent Ca 2+ flux may contribute to the heart’s adaptations to chronic stress. We therefore compared mice with adult cardiomyocyte (ACM)-specific loss ( Mcu fl/fl ; Mcu-cKO) or gain (CAG-CAT-MCU; MCU-Tg) of MCU function to examine the role of MCU-dependent m Ca 2+ uptake in a model of chronic catecholamine overload. In vitro characterization of ACMs confirmed that MCU overexpression enhanced and Mcu deletion inhibited acute m Ca 2+ uptake. Neither loss nor gain of MCU function altered baseline contractile function in vivo . In αMHC-MCM control mice, fractional shortening was transiently increased after 2 days of isoproterenol infusion. This initial increase in contractility was attenuated in MCU-cKO mice. In contrast, MCU-Tg mice exhibited decreased fractional shortening at 7 and 14 days of isoproterenol infusion. This detrimental effect on contractile function was associated with increased LV dilation, HW/BW ratio, and lung edema. MCU-Tg cardiomyocytes in vitro exhibited increased ROS production and a trend towards increased cell death upon elevation of cytosolic Ca 2+ with ionomycin. These data prompted us to test the hypothesis that isoproterenol-induced contractile dysfunction in MCU-Tg hearts is caused by cardiomyocyte dropout due to m Ca 2+ overload and mitochondrial permeability transition. However, genetic deletion of the mPTP component cyclophilin D did not prevent the decline in contractile function, diminish cardiomyocyte death, or attenuate LV remodeling in MCU-Tg animals during chronic isoproterenol infusion. We conclude that although mtCU-dependent m Ca 2+ uptake is essential for early energetic adaptations to high adrenergic load, under conditions of chronic adrenergic stress it is maladaptive and predisposes to heart failure. Our data suggest that this maladaptive response occurs via mechanisms independent of cyclophilin D-mediated permeability transition.

The adult mammalian heart has a limited regenerative capacity. Therefore, identification of endogenous cells and mechanisms that contribute to cardiac regeneration is essential for the development of targeted therapies. The side population (SP) phenotype has been used to enrich for stem cells throughout the body; however, SP cells isolated from the heart have been studied exclusively in cell culture or after transplantation, limiting our understanding of their function in vivo. We generated a new Abcg2-driven lineage-tracing mouse model with efficient labeling of SP cells. Labeled SP cells give rise to terminally differentiated cells in bone marrow and intestines. In the heart, labeled SP cells give rise to lineage-traced cardiomyocytes under homeostatic conditions with an increase in this contribution following cardiac injury. Instead of differentiating into cardiomyocytes like proposed cardiac progenitor cells, cardiac SP cells fuse with preexisting cardiomyocytes to stimulate cardiomyocyte cell cycle reentry. Our study is the first to show that fusion between cardiomyocytes and non-cardiomyocytes, identified by the SP phenotype, contribute to endogenous cardiac regeneration by triggering cardiomyocyte cell cycle reentry in the adult mammalian heart.

Calcium (Ca 2+ ) uptake into the mitochondrial matrix occurs via the mitochondrial Ca 2+ uniporter channel (mtCU) and tunes mitochondrial metabolism to meet acute changes in cellular ATP demand. However, the role of mtCU-dependent mitochondrial Ca 2+ ( m Ca 2+ ) uptake in regulating homeostatic heart function and adaptation to chronic increases in workload remain controversial. We subjected mice with tamoxifen-inducible, cardiomyocyte-specific gain (flox-stop-MCU x αMHC-MCM, MCU-Tg) or loss ( Mcu fl/fl x αMHC-MCM , Mcu- cKO) of MCU function to 2wk isoproterenol (iso) infusion to test the hypothesis that m Ca 2+ uptake through MCU contributes to functional adaptation to sustained catecholamine signaling. Neither gain nor loss of MCU function altered baseline cardiac structure or function. Fractional shortening was increased after 2d of iso infusion in MCM control mice, but loss of MCU blocked this effect. In contrast, fractional shortening declined significantly after 7-14d of iso in MCU-Tg mice. MCU-Tg mice also exhibited increased LV dilation, heart mass, and lung edema compared to controls after 14d of iso. Acute treatment of MCU-Tg cardiomyocytes in vitro with the Ca 2+ ionophore ionomycin revealed increased ROS production and a trend towards increased sensitivity to Ca 2+ -induced cell death. In agreement, Evans blue dye exclusion assays revealed increased cardiomyocyte necrosis in MCU-Tg hearts following iso. Therefore, we deleted the mPTP regulator cyclophilin D (CypD) in MCU-Tg mice to test whether cardiomyocyte dropout due to m Ca 2+ overload-induced mPTP contributed to iso-induced contractile dysfunction. Unexpectedly, CypD deletion failed to attenuate contractile dysfunction and hypertrophic remodeling, and increased rather than attenuated iso-induced cardiomyocyte death in MCU-Tg mice. We conclude that while MCU-dependent m Ca 2+ uptake is required for the heart’s initial contractile response to catecholaminergic stress, under prolonged stimulation augmented m Ca 2+ uptake becomes deleterious and predisposes to cardiomyocyte death and heart failure. Our findings support the notion that the detrimental effects of sustained MCU-dependent m Ca 2+ uptake are mediated by mechanisms distinct from mitochondrial permeability transition.

KEYWORDS: Calcium channelscalcium signalingMCUBMCUmitochondriamitochondrial permeability transitionischemia-reperfusion injuryheartcalcium uptake

‘Lokiarchaea’, previously known only from DNA, is isolated and grown in culture.

A firm hired by the National Institutes of Health will work with participants in a research programme that plans to sequence one million genomes. A firm hired by the National Institutes of Health will work with participants in a research programme that plans to sequence one million genomes.

Impairments in neuronal intracellular calcium ( i Ca 2+ ) handling may contribute to Alzheimer’s disease (AD) development. Metabolic dysfunction and progressive neuronal loss are associated with AD progression, and mitochondrial calcium ( m Ca 2+ ) signaling is a key regulator of both of these processes. Here, we report remodeling of the m Ca 2+ exchange machinery in the prefrontal cortex of individuals with AD. In the 3xTg-AD mouse model impaired m Ca 2+ efflux capacity precedes neuropathology. Neuronal deletion of the mitochondrial Na + /Ca 2+ exchanger (NCLX, Slc8b1 gene) accelerated memory decline and increased amyloidosis and tau pathology. Further, genetic rescue of neuronal NCLX in 3xTg-AD mice is sufficient to impede AD-associated pathology and memory loss. We show that m Ca 2+ overload contributes to AD progression by promoting superoxide generation, metabolic dysfunction and neuronal cell death. These results provide a link between the calcium dysregulation and metabolic dysfunction hypotheses of AD and suggest m Ca 2+ exchange as potential therapeutic target in AD.

Necrotic cell death is the main way in which cells die during myocardial infarction (MI) and heart failure (HF), yet the molecular mechanisms regulating necrotic cell death are poorly defined. To elucidate the key regulators of plasma membrane rupture during necrotic cell death a genome-wide shRNA loss-of-function screen was performed which identified components of SNARE-mediated membrane fusion as potential facilitators of Ca 2+ and ROS-induced necrosis. To examine if the SNARE machinery is involved in cellular necrosis we targeted N-ethylmaleimide sensitive Factor (NSF) due to its requirement in SNARE recycling, lack of gene homologs, and redox sensitivity. Deletion of NSF from 3T3 fibroblasts by CRISPR-Cas9n (Nsf -/- ), inhibited membrane rupture and improved cell viability following Ca 2+ overload (ionomycin), ROS (H 2 O 2 ), and necroptotic (TNFa, CHX, zVAD)-induced cell death but did not alter apoptotic (staurosporine) cellular demise. We next created a cardiac-specific conditional Nsf knockout mouse model to determine if NSF contributes to myocyte death during IR injury. Loss of NSF in cardiomyocytes did not alter baseline cardiac function or structure. Currently, studies are underway to determine if NSF contributes to necrotic cell as occurs in IR injury and heart failure. In summary, our results suggest that NSF is an important molecular component of membrane rupture and pathogenic cell death.

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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.