EMRE is essential for mitochondrial calcium uniporter activity in a mouse model.

The mitochondrial calcium uniporter is widely accepted as the primary route of rapid calcium entry into mitochondria, where increases in matrix calcium contribute to bioenergetics but also mitochondrial permeability and cell death. Hence, regulation of uniporter activity is critical to mitochondrial homeostasis. The uniporter subunit EMRE is known to be an essential regulator of the channel-forming protein MCU in cell culture, but EMRE's impact on organismal physiology is less understood. Here we characterize a mouse model of EMRE deletion and show that EMRE is indeed required for mitochondrial calcium uniporter function in vivo. EMRE-/- mice are born less frequently; however, the mice that are born are viable, healthy, and do not manifest overt metabolic impairment, at rest or with exercise. Finally, to investigate the role of EMRE in disease processes, we examine the effects of EMRE deletion in a muscular dystrophy model associated with mitochondrial calcium overload.

Cyclophilin D-mediated regulation of the permeability transition pore is altered in mice lacking the mitochondrial calcium uniporter.

Aims: Knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice abrogates mitochondrial Ca2+ uptake and permeability transition pore (PTP) opening. However, hearts from global MCU-KO mice are not protected from ischaemic injury. We aimed to investigate whether adaptive alterations occur in cell death signalling pathways in the hearts of global MCU-KO mice. Methods and results: First, we examined whether cell death may occur via an upregulation in necroptosis in MCU-KO mice. However, our results show that neither RIP1 inhibition nor RIP3 knockout afford protection against ischaemia-reperfusion injury in MCU-KO as in wildtype (WT) hearts, indicating that the lack of protection cannot be explained by upregulation of necroptosis. Instead, we have identified alterations in cyclophilin D (CypD) signalling in MCU-KO hearts. In the presence of a calcium ionophore, MCU-KO mitochondria take up calcium and do undergo PTP opening. Furthermore, PTP opening in MCU-KO mitochondria has a lower calcium retention capacity (CRC), suggesting that the calcium sensitivity of PTP is higher. Phosphoproteomics identified an increase in phosphorylation of CypD-S42 in MCU-KO. We investigated the interaction of CypD with the putative PTP component ATP synthase and identified an approximately 50% increase in this interaction in MCU-KO cardiac mitochondria. Mutation of the novel CypD phosphorylation site S42 to a phosphomimic reduced CRC, increased CypD-ATP synthase interaction by approximately 50%, and increased cell death in comparison to a phospho-resistant mutant. Conclusion: Taken together these data suggest that MCU-KO mitochondria exhibit an increase in phosphorylation of CypD-S42 which decreases PTP calcium sensitivity thus allowing activation of PTP in the absence of an MCU-mediated increase in matrix calcium.

Mitochondrial Permeability Transition Pore and Calcium Handling.

The opening of a large conductance channel in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore (PTP), has been shown to be a primary mediator of cell death in the heart subjected to ischemia-reperfusion injury. Inhibitors of the PTP have been shown to reduce cardiac ischemia-reperfusion injury in many animal models. Furthermore, most cardioprotective strategies appear to reduce ischemic cell death either by reducing the triggers for the opening of the PTP, such as reducing calcium overload or reactive oxygen species, or by inhibiting PTP modulators. This chapter will focus on key issues in the study of the PTP and provide some methods for measuring PTP opening in isolated mitochondria.

The impact of ovariectomy on cardiac excitation-contraction coupling is mediated through cAMP/PKA-dependent mechanisms.

Ovariectomy (OVX) promotes sarcoplasmic reticulum (SR) Ca2+ overload in ventricular myocytes. We hypothesized that the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway contributes to this Ca2+ dysregulation. Myocytes were isolated from adult female C57BL/6 mice following either OVX or sham surgery (surgery at ≈1mos). Contractions, Ca2+ concentrations (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in voltage-clamped myocytes. Intracellular cAMP levels were determined with an enzyme immunoassay; phosphodiesterase (PDE) and adenylyl cyclase (AC) isoform expression was examined with qPCR. Ca2+ currents were similar in myocytes from sham and OVX mice but Ca2+ transients, excitation-contraction (EC)-coupling gain, SR content and contractions were larger in OVX than sham cells. To determine if the cAMP/PKA pathway mediated OVX-induced alterations in EC-coupling, cardiomyocytes were incubated with the PKA inhibitor H-89 (2µM), which abolished baseline differences. While basal intracellular cAMP did not differ, levels were higher in OVX than sham in the presence of a non-selective PDE inhibitor (300µM IBMX), or an AC activator (10µM forskolin). This suggests the production of cAMP by AC and its breakdown by PDE were enhanced by OVX. Consistent with this, mRNA levels for both AC5 and PDE4A were higher in OVX in comparison to sham. Differences in Ca2+ homeostasis and contractions were abolished when sham and OVX cells were dialyzed with patch pipettes containing the same concentration of 8-bromoadenosine-cAMP (50µM). Interestingly, selective inhibition of PDE4 increased Ca2+ current only in OVX cells. Together, these findings suggest that estrogen suppresses SR Ca2+ release and that this is regulated, at least in part, by the cAMP/PKA pathway. These changes in the cAMP/PKA pathway may promote Ca2+ dysregulation and cardiovascular disease when ovarian estrogen levels fall. These results advance our understanding of female-specific cardiomyocyte mechanisms that may affect responses to therapeutic interventions in older women.

The In Vivo Biology of the Mitochondrial Calcium Uniporter.

The identification of the molecular composition of the mitochondrial calcium uniporter has allowed for the genetic manipulation of its components and the creation of various in vivo genetic models. Here, we review the initial attempts to modulate the expression of components of the calcium uniporter in a range of organisms from plants to mammals. This analysis has confirmed the strict requirement for the uniporter for in vivo mitochondrial calcium uptake and for maintaining mitochondrial calcium homeostasis. We further discuss the physiological effects following genetic manipulation of the uniporter on tissue bioenergetics and the threshold for cell death. Finally, we analyze the limited information regarding the role of various uniporter components in human disease.

Sex Differences in Metabolic Cardiomyopathy.

In contrast to ischemic cardiomyopathies which are more common in men, women are over-represented in diabetic cardiomyopathies. Diabetes is a risk factor for cardiovascular disease; however, there is a sexual dimorphism in this risk factor: heart disease is five times more common in diabetic women but only two-times more common in diabetic men. Heart failure with preserved ejection fraction, which is associated with metabolic syndrome, is also more prevalent in women. This review will examine potential mechanisms for the sex differences in metabolic cardiomyopathies. Sex differences in metabolism, calcium handling, nitric oxide, and structural proteins will be evaluated. Nitric oxide synthase and PPARα exhibit sex differences and have also been proposed to mediate the development of hypertrophy and heart failure. We focused on a role for these signalling pathways in regulating sex differences in metabolic cardiomyopathies.

MICU1 Serves as a Molecular Gatekeeper to Prevent In Vivo Mitochondrial Calcium Overload.

MICU1 is a component of the mitochondrial calcium uniporter, a multiprotein complex that also includes MICU2, MCU, and EMRE. Here, we describe a mouse model of MICU1 deficiency. MICU1(-/-) mitochondria demonstrate altered calcium uptake, and deletion of MICU1 results in significant, but not complete, perinatal mortality. Similar to afflicted patients, viable MICU1(-/-) mice manifest marked ataxia and muscle weakness. Early in life, these animals display a range of biochemical abnormalities, including increased resting mitochondrial calcium levels, altered mitochondrial morphology, and reduced ATP. Older MICU1(-/-) mice show marked, spontaneous improvement coincident with improved mitochondrial calcium handling and an age-dependent reduction in EMRE expression. Remarkably, deleting one allele of EMRE helps normalize calcium uptake while simultaneously rescuing the high perinatal mortality observed in young MICU1(-/-) mice. Together, these results demonstrate that MICU1 serves as a molecular gatekeeper preventing calcium overload and suggests that modulating the calcium uniporter could have widespread therapeutic benefits.

Assessment of cardiac function in mice lacking the mitochondrial calcium uniporter.

Mitochondrial calcium is thought to play an important role in the regulation of cardiac bioenergetics and function. The entry of calcium into the mitochondrial matrix requires that the divalent cation pass through the inner mitochondrial membrane via a specialized pore known as the mitochondrial calcium uniporter (MCU). Here, we use mice deficient of MCU expression to rigorously assess the role of mitochondrial calcium in cardiac function. Mitochondria isolated from MCU(-/-) mice have reduced matrix calcium levels, impaired calcium uptake and a defect in calcium-stimulated respiration. Nonetheless, we find that the absence of MCU expression does not affect basal cardiac function at either 12 or 20months of age. Moreover, the physiological response of MCU(-/-) mice to isoproterenol challenge or transverse aortic constriction appears similar to control mice. Thus, while mitochondria derived from MCU(-/-) mice have markedly impaired mitochondrial calcium handling, the hearts of these animals surprisingly appear to function relatively normally under basal conditions and during stress.

The ins and outs of mitochondrial calcium.

Calcium is thought to play an important role in regulating mitochondrial function. Evidence suggests that an increase in mitochondrial calcium can augment ATP production by altering the activity of calcium-sensitive mitochondrial matrix enzymes. In contrast, the entry of large amounts of mitochondrial calcium in the setting of ischemia-reperfusion injury is thought to be a critical event in triggering cellular necrosis. For many decades, the details of how calcium entered the mitochondria remained a biological mystery. In the past few years, significant progress has been made in identifying the molecular components of the mitochondrial calcium uniporter complex. Here, we review how calcium enters and leaves the mitochondria, the growing insight into the topology, stoichiometry and function of the uniporter complex, and the early lessons learned from some initial mouse models that genetically perturb mitochondrial calcium homeostasis.

Sex differences in SR Ca(2+) release in murine ventricular myocytes are regulated by the cAMP/PKA pathway.

Previous studies have shown that ventricular myocytes from female rats have smaller contractions and Ca(2+) transients than males. As cardiac contraction is regulated by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway, we hypothesized that sex differences in cAMP contribute to differences in Ca(2+) handling. Ca(2+) transients (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in ventricular myocytes from adult male and female C57BL/6 mice. Under basal conditions, diastolic Ca(2+), sarcoplasmic reticulum (SR) Ca(2+) stores, and L-type Ca(2+) current did not differ between the sexes. However, female myocytes had smaller Ca(2+) transients (26% smaller), Ca(2+) sparks (6% smaller), and excitation-contraction coupling gain in comparison to males (23% smaller). Interestingly, basal levels of intracellular cAMP were lower in female myocytes (0.7±0.1 vs. 1.7±0.2fmol/µg protein; p<0.001). Importantly, PKA inhibition (2µM H-89) eliminated male-female differences in Ca(2+) transients and gain, as well as Ca(2+) spark amplitude. Western blots showed that PKA inhibition also reduced the ratio of phospho:total RyR2 in male hearts, but not in female hearts. Stimulation of cAMP production with 10µM forskolin abolished sex differences in cAMP levels, as well as differences in Ca(2+) transients, sparks, and gain. To determine if the breakdown of cAMP differed between the sexes, phosphodiesterase (PDE) mRNA levels were measured. PDE3 expression was similar in males and females, but PDE4B expression was higher in female ventricles. The inhibition of cAMP breakdown by PDE4 (10µM rolipram) abolished differences in Ca(2+) transients and gain. These findings suggest that female myocytes have lower levels of basal cAMP due, in part, to higher expression of PDE4B. Lower cAMP levels in females may attenuate PKA phosphorylation of Ca(2+) handling proteins in females, and may limit positive inotropic responses to stimulation of the cAMP/PKA pathway in female hearts.

Sex differences in mechanisms of cardiac excitation-contraction coupling.

The incidence and expression of cardiovascular diseases differs between the sexes. This is not surprising, as cardiac physiology differs between men and women. Clinical and basic science investigations have shown important sex differences in cardiac structure and function. The pervasiveness of sex differences suggests that such differences must be fundamental, likely operating at a cellular level. Indeed, studies have shown that isolated ventricular myocytes from female animals have smaller and slower contractions and underlying calcium transients compared to males. Recent evidence suggests that this arises from sex differences in components of the cardiac excitation-contraction coupling pathway, the sequence of events linking myocyte depolarization to calcium release from the sarcoplasmic reticulum and subsequent contraction. The concept that sex hormones may regulate intracellular calcium at the level of the cardiomyocyte is important, as levels of these hormones decline in both men and women as the incidence of cardiovascular disease rises. This review focuses on the impact of sex on cardiac contraction, in particular at the cellular level, and highlights specific components of the excitation-contraction coupling pathway that differ between the sexes. Understanding sex hormone regulation of calcium homeostasis in the heart may reveal new avenues for therapeutic strategies to treat cardiac dysfunction and cardiovascular diseases.

H-89 decreases the gain of excitation-contraction coupling and attenuates calcium sparks in the absence of beta-adrenergic stimulation.

This study used the selective protein kinase A (PKA) inhibitor H-89 (N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) to determine the role of basal PKA activity in modulating cardiac excitation-contraction coupling in the absence of ß-adrenergic stimulation. Basal intracellular cyclic AMP (cAMP) levels measured in isolated murine ventricular myocytes with an enzyme immunoassay were increased upon adenylyl cyclase activation (forskolin; 1 and 10 µM) or phosphodiesterase inhibition (3-isobutyl-1-methylxanthine, IBMX; 300 µM). Forskolin and IBMX also caused concentration-dependent increases in peak Ca(2+) transients (fura-2) and cell shortening (edge-detector) measured simultaneously in field-stimulated myocytes (37 °C). Similar effects were seen upon application of dibutyryl cAMP. In voltage-clamped myocytes, H-89 (2 µM) decreased basal Ca(2+) transients, contractions and underlying Ca(2+) currents. H-89 also decreased diastolic Ca(2+) and the gain of excitation-contraction coupling (Ca(2+) release/Ca(2+) current), especially at negative membrane potentials. This was independent of alterations in sarcoplasmic reticulum (SR) Ca(2+) loading, as SR stores were unchanged by PKA inhibition. H-89 also decreased the frequency, amplitude and width of spontaneous Ca(2+) sparks measured in quiescent myocytes (loaded with fluo-4), but increased time-to-peak. Thus, H-89 suppressed SR Ca(2+) release by decreasing Ca(2+) current and by reducing the gain of excitation-contraction coupling, in part by decreasing the size of individual Ca(2+) release units. These data suggest that basal PKA activity enhances SR Ca(2+) release in the absence of ß-adrenergic stimulation. This may depress contractile function in models such as aging, where the cAMP/PKA pathway is altered due to low basal cAMP levels.

Ovariectomy enhances SR Ca²âº release and increases Ca²âº spark amplitudes in isolated ventricular myocytes.

This study compared Ca(2+) homeostasis in ventricular myocytes from 8-month-old female C57BL/6J mice that had either a bilateral ovariectomy (OVX) or a sham surgery at 3 weeks of age. Cells were loaded with fura-2 and field-stimulated or voltage-clamped with steps to membrane potentials between -40 and +80 mV (37°C). Peak Ca(2+) transients increased by two-fold in OVX myocytes when compared to sham, and Ca(2+) transient rates of rise and decay were faster in OVX cells. In contrast, Ca(2+) current densities were similar in sham and OVX cells. Sarcoplasmic reticulum (SR) Ca(2+) content, assessed by caffeine, also was higher in OVX compared to sham cells (111.7 ± 11.9 vs. 61.2 ± 10.4 nM; p<0.05). Furthermore, the gain of Ca(2+) release (Ca(2+) release/Ca(2+) current) was significantly greater in OVX than in sham cells (16.3 ± 2.5 vs. 7.7 ± 2.0 nM/pApF(-1) at 0 mV; p<0.05). As changes in unitary Ca(2+) release might account for the increased gain in OVX cells, spontaneous Ca(2+) sparks were compared in fluo-4-loaded myocytes (37°C). Spark frequency was higher in OVX cells than in sham cells. In addition, spark amplitudes were greater in OVX than in sham myocytes (ΔF/F(0)=0.379 ± 0.006 vs. 0.342 ± 0.006; p<0.05). However, spark widths and time courses were similar in the two groups. These data suggest that the size of individual SR Ca(2+) release units is larger and the SR Ca(2+) content is greater in myocytes of OVX mice, producing augmented gain and SR Ca(2+) release. These observations show that OVX disrupts intracellular Ca(2+) homeostasis and suggest that sex steroid hormones modulate unitary Ca(2+) release in individual cardiac myocytes.

A procedure for creating a frailty index based on deficit accumulation in aging mice.

This study developed an approach to quantify frailty with a frailty index (FI) and investigated whether age-related changes in contractions, calcium transients, and ventricular myocyte length were more prominent in mice with a high FI. The FI combined 31 variables that reflect different aspects of health in middle-aged (∼12 months) and aged (∼30 months) mice of both sexes. Aged animals had a higher FI than younger animals (FI = 0.43 ± 0.03 vs 0.08 ± 0.02, p < .001, n = 12). Myocyte hypertrophy increased by 30%-50% as the FI increased in aged animals. Peak contractions decreased more than threefold from lowest to highest FI values in aged mice (p < .037), but calcium transients were unaffected. Similar results were seen with an FI based on eight noninvasive variables identified as underlying factors. These results show that an FI can be developed for murine models and suggest that age-associated changes in myocytes are more prominent in animals with a high FI.