A digitized-fluorescence-imaging study of mitochondrial Ca2+ increase by doxorubicin in cardiac myocytes.
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ABSTRACT:
SUBMITTER: Chacon E
PROVIDER: S-EPMC1130770 | biostudies-other | 1992 Feb
REPOSITORIES: biostudies-other
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Project description:RATIONALE:Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes. OBJECTIVE:To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes. METHODS AND RESULTS:Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy. CONCLUSIONS:Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.
| S-EPMC4740258 | biostudies-literature
Project description:Metabolic inhibition is a common condition observed during ischemic heart disease and heart failure. It is usually accompanied by a reduction in L-type Ca2+ channel (LTCC) activity. In this study, however, we show that metabolic inhibition results in a biphasic effect on LTCC current (ICaL) in human and rat cardiac myocytes: an initial increase of ICaL is observed in the early phase of metabolic inhibition which is followed by the more classical and strong inhibition. We studied the mechanism of the initial increase of ICaL in cardiac myocytes during β-adrenergic stimulation by isoprenaline, a non-selective agonist of β-adrenergic receptors. The whole-cell patch⁻clamp technique was used to record the ICaL in single cardiac myocytes. The initial increase of ICaL was induced by a wide range of metabolic inhibitors (FCCP, 2,4-DNP, rotenone, antimycin A). In rat cardiomyocytes, the initial increase of ICaL was eliminated when the cells were pre-treated with thapsigargin leading to the depletion of Ca2+ from the sarcoplasmic reticulum (SR). Similar results were obtained when Ca2+ release from the SR was blocked with ryanodine. These data suggest that the increase of ICaL in the early phase of metabolic inhibition is due to a reduced calcium dependent inactivation (CDI) of LTCCs. This was further confirmed in human atrial myocytes where FCCP failed to induce the initial stimulation of ICaL when Ca2+ was replaced by Ba2+, eliminating CDI of LTCCs. We conclude that the initial increase in ICaL observed during the metabolic inhibition in human and rat cardiomyocytes is a consequence of an acute reduction of Ca2+ release from SR resulting in reduced CDI of LTCCs.
| S-EPMC6471217 | biostudies-literature
Project description:Mitochondrial Ca2+ ([Ca2+]m) regulates oxidative phosphorylation and thus contributes to energy supply and demand matching in cardiac myocytes. Mitochondria take up Ca2+ via the Ca2+ uniporter (MCU) and extrude it through the mitochondrial Na+/Ca2+ exchanger (mNCE). It is controversial whether mitochondria take up Ca2+ rapidly, on a beat-to-beat basis, or slowly, by temporally integrating cytosolic Ca2+ ([Ca2+]c) transients. Furthermore, although mitochondrial Ca2+ efflux is governed by mNCE, it is unknown whether elevated intracellular Na+ ([Na+]i) affects mitochondrial Ca2+ uptake and bioenergetics. To monitor [Ca2+]m, mitochondria of guinea pig cardiac myocytes were loaded with rhod-2-acetoxymethyl ester (rhod-2 AM), and [Ca2+]c was monitored with indo-1 after dialyzing rhod-2 out of the cytoplasm. [Ca2+]c transients, elicited by voltage-clamp depolarizations, were accompanied by fast [Ca2+]m transients, whose amplitude (delta) correlated linearly with delta[Ca2+]c. Under beta-adrenergic stimulation, [Ca2+]m decay was approximately 2.5-fold slower than that of [Ca2+]c, leading to diastolic accumulation of [Ca2+]m when amplitude or frequency of delta[Ca2+]c increased. The MCU blocker Ru360 reduced delta[Ca2+]m and increased delta[Ca2+]c, whereas the mNCE inhibitor CGP-37157 potentiated diastolic [Ca2+]m accumulation. Elevating [Na+]i from 5 to 15 mmol/L accelerated mitochondrial Ca2+ decay, thus decreasing systolic and diastolic [Ca2+]m. In response to gradual or abrupt changes of workload, reduced nicotinamide-adenine dinucleotide (NADH) levels were maintained at 5 mmol/L [Na+]i, but at 15 mmol/L, the NADH pool was partially oxidized. The results indicate that (1) mitochondria take up Ca2+ rapidly and contribute to fast buffering during a [Ca2+]c transient; and (2) elevated [Na+]i impairs mitochondrial Ca2+ uptake, with consequent effects on energy supply and demand matching. The latter effect may have implications for cardiac diseases with elevated [Na+]i.
| S-EPMC2711867 | biostudies-literature
Project description:In cardiomyocytes, Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs) binds to and activates RyR2 channels, resulting in subsequent Ca2+ release from the sarcoplasmic reticulum (SR) and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca2+-activated K+ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca2+ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca2+-activated K+ current (IK,Ca) recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) (p<0.05, p<0.01, respectively). The activation of RyR2 by caffeine increased the IK,Ca in the cardiac cells (p<0.05, p<0.01, respectively). We further analyzed the effect of RyR2 knockdown on IK,Ca and Ca2+ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in IK,Ca (p<0.05) and [Ca2+]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca2+ release is responsible for the activation and modulation of SK channels in cardiac myocytes.
| S-EPMC3991633 | biostudies-literature
Project description:SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.
| S-EPMC5855641 | biostudies-literature
Project description:Insulin inhibits ischemia/reperfusion-induced myocardial apoptosis through the PI3K/Akt/mTOR pathway. Survivin is a key regulator of anti-apoptosis against doxorubicin-induced cardiotoxicity. Insulin increases survivin expression in cardiac myocytes to mediate cytoprotection. However, the mechanism by which survivin mediates the protective effect of insulin against doxorubicin-associated injury remains to be determined. In this study, we demonstrated that pretreatment of H9c2 cardiac myocytes with insulin resulted in a significant decrease in doxorubicin-induced apoptotic cell death by reducing cytochrome c release and caspase-3 activation. Doxorubicin-induced reduction of survivin mRNA and protein levels was also significantly perturbed by insulin pretreatment. Reducing survivin expression with survivin siRNA abrogated insulin-mediated inhibition of caspase-3 activation, suggesting that insulin signals to survivin inhibited caspase-3 activation. Interestingly, pretreatment of H9c2 cells with insulin or MG132, a proteasome inhibitor, inhibited doxorubicin-induced degradation of the transcription factor Sp1. ChIP assay showed that pretreatment with insulin inhibited doxorubicin-stimulated Sp1 dissociation from the survivin promoter. Finally using pharmacological inhibitors of the PI3K pathway, we showed that insulin-mediated activation of the PI3K/Akt/mTORC1 pathway prevented doxorubicin-induced proteasome-mediated degradation of Sp1. Taken together, insulin pretreatment confers a protective effect against doxorubicin-induced cardiotoxicity by promoting Sp1-mediated transactivation of survivin to inhibit apoptosis. Our study is the first to define a role for survivin in cellular protection by insulin against doxorubicin-associated injury and show that Sp1 is a critical factor in the transcriptional regulation of survivin.
| S-EPMC4535909 | biostudies-literature
Project description:In ventricular myocardium, extracellular matrix (ECM) remodeling is a hallmark of physiological and pathological growth, coincident with metabolic rewiring of cardiac myocytes. However, the direct impact of the biochemical and mechanical properties of the ECM on the metabolic function of cardiac myocytes is mostly unknown. Furthermore, understanding the impact of distinct biomaterials on cardiac myocyte metabolism is critical for engineering physiologically-relevant models of healthy and diseased myocardium. For these reasons, we systematically measured morphological and metabolic responses of neonatal rat ventricular myocytes cultured on fibronectin- or gelatin-coated polydimethylsiloxane (PDMS) of three elastic moduli and gelatin hydrogels with four elastic moduli. On all substrates, total protein content, cell morphology, and the ratio of mitochondrial DNA to nuclear DNA were preserved. Cytotoxicity was low on all substrates, although slightly higher on PDMS compared to gelatin hydrogels. We also quantified oxygen consumption rates and extracellular acidification rates using a Seahorse extracellular flux analyzer. Our data indicate that several metrics associated with baseline glycolysis and baseline and maximum mitochondrial function are enhanced when cardiac myocytes are cultured on gelatin hydrogels compared to all PDMS substrates, irrespective of substrate rigidity. These results yield new insights into how mechanical and biochemical cues provided by the ECM impact mitochondrial function in cardiac myocytes. STATEMENT OF SIGNIFICANCE: Cardiac development and disease are associated with remodeling of the extracellular matrix coincident with metabolic rewiring of cardiac myocytes. However, little is known about the direct impact of the biochemical and mechanical properties of the extracellular matrix on the metabolic function of cardiac myocytes. In this study, oxygen consumption rates were measured in neonatal rat ventricular myocytes maintained on several commonly-used biomaterial substrates to reveal new relationships between the extracellular matrix and cardiac myocyte metabolism. Several mitochondrial parameters were enhanced on gelatin hydrogels compared to synthetic PDMS substrates. These data are important for comprehensively understanding matrix-regulation of cardiac myocyte physiology. Additionally, these data should be considered when selecting scaffolds for engineering in vitro cardiac tissue models.
| S-EPMC6801042 | biostudies-literature
Project description:Compartmentation of cAMP signaling is a critical factor for maintaining the integrity of receptor-specific responses in cardiac myocytes. This phenomenon relies on various factors limiting cAMP diffusion. Our previous work in adult rat ventricular myocytes (ARVMs) indicates that PKA regulatory subunits anchored to the outer membrane of mitochondria play a key role in buffering the movement of cytosolic cAMP. PKA can be targeted to discrete subcellular locations through the interaction of both type I and type II regulatory subunits with A-kinase anchoring proteins (AKAPs). The purpose of this study is to identify which AKAPs and PKA regulatory subunit isoforms are associated with mitochondria in ARVMs. Quantitative PCR data demonstrate that mRNA for dual specific AKAP1 and 2 (D-AKAP1 & D-AKAP2), acyl-CoA-binding domain-containing 3 (ACBD3), optic atrophy 1 (OPA1) are most abundant, while Rab32, WAVE-1, and sphingosine kinase type 1 interacting protein (SPHKAP) were barely detectable. Biochemical and immunocytochemical analysis suggests that D-AKAP1, D-AKAP2, and ACBD3 are the predominant mitochondrial AKAPs exposed to the cytosolic compartment in these cells. Furthermore, we show that both type I and type II regulatory subunits of PKA are associated with mitochondria. Taken together, these data suggest that D-AKAP1, D-AKAP2, and ACBD3 may be responsible for tethering both type I and type II PKA regulatory subunits to the outer mitochondrial membrane in ARVMs. In addition to regulating PKA-dependent mitochondrial function, these AKAPs may play an important role by buffering the movement of cAMP necessary for compartmentation.
| S-EPMC8436057 | biostudies-literature
Project description:Oscillatory behavior of mitochondrial inner membrane potential (ΔΨm) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨm depolarization as well as synchronized limit cycle oscillations of ΔΨm in the network. The functional impact of ΔΨm instability on cardiac electrophysiology, Ca(2+) handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨm oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10(-2) ± 0.98 × 10(-2) s(-1)) and pyruvate (7.49 × 10(-2) ± 1.65 × 10(-2) s(-1)) than lactate (4.83 × 10(-2) ± 1.25 × 10(-2) s(-1)) or β-hydroxybutyrate (4.11 × 10(-2) ± 0.62 × 10(-2) s(-1)). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨm instability, ultimately scaling to produce organ level dysfunction.
| S-EPMC4407245 | biostudies-literature
Project description:Mitochondria are involved in multiple cellular functions, in addition to their core role in energy metabolism. Mitochondria localized in different cellular locations may have different morphology, Ca2+ handling and biochemical properties and may interact differently with other intracellular structures, causing functional specificity. However, most prior studies have utilized isolated mitochondria, removed from their intracellular environment. Mitochondria in cardiac ventricular myocytes are highly organized, with a majority squeezed between the myofilaments in longitudinal chains (intrafibrillar mitochondria, IFM). There is another population of perinuclear mitochondria (PNM) around and between the two nuclei typical in myocytes. Here, we take advantage of live myocyte imaging to test for quantitative morphological and functional differences between IFM and PNM with respect to calcium fluxes, membrane potential, sensitivity to oxidative stress, shape and dynamics. Our findings show higher mitochondrial Ca2+ uptake and oxidative stress sensitivity for IFM vs. PNM, which may relate to higher local energy demand supporting the contractile machinery. In contrast to IFM which are remarkably static, PNM are relatively mobile, appear to participate readily in fission/fusion dynamics and appear to play a central role in mitochondrial genesis and turnover. We conclude that while IFM may be physiologically tuned to support local myofilament energy demands, PNM may be more critical in mitochondrial turnover and regulation of nuclear function and import/export. Thus, important functional differences are present in intrafibrillar vs. perinuclear mitochondrial subpopulations.
| S-EPMC7173146 | biostudies-literature