Fatty acid (FA)-dependent oxidation is the predominant process for energy supply in normal heart. Impaired FA metabolism and metabolic insufficiency underlie the failing of the myocardium. So far, FA metabolism in normal cardiac physiology and heart failure remains undetermined. Here, we evaluate the mechanisms of FA and major metabolic substrates (termed NF) on the contraction, relaxation, and Ca2+ handling in rat left ventricular (LV) myocytes. Our results showed that NF significantly increased myocyte contraction and facilitated relaxation. Moreover, NF increased the amplitudes of diastolic and systolic Ca2+ transients ([Ca2+]i), abbreviated time constant of [Ca2+]i decay (tau), and prolonged the peak duration of [Ca2+]i. Whole-cell patch-clamp experiments revealed that NF increased Ca2+ influx via L-type Ca2+ channels (LTCC, ICa-integral) and prolonged the action potential duration (APD). Further analysis revealed that NF shifted the relaxation phase of sarcomere lengthening vs. [Ca2+]i trajectory to the right and increased [Ca2+]i for 50 % of sarcomere relengthening (EC50), suggesting myofilament Ca2+ desensitization. Butanedione monoxime (BDM), a myosin ATPase inhibitor that reduces myofilament Ca2+ sensitivity, abolished the NF-induced enhancement of [Ca2+]i amplitude and the tau of [Ca2+]i decay, indicating the association of myofilament Ca2+ desensitization with the changes in [Ca2+]i profile in NF. NF reduced intracellular pH ([pHi]). Increasing [pH]i buffer capacity with HCO3/CO2 attenuated Δ [pH]i and reversed myofilament Ca2+ desensitization and Ca2+ handling in NF. Collectively, greater Ca2+ influx through LTCCs and myofilament Ca2+ desensitization, via reducing [pH]i, are likely responsible for the positive inotropic and lusitropic effects of NF. Computer simulation recapitulated the effects of NF.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-016-1892-8) contains supplementary material, which is available to authorized users.
Introduction: COVID-19 patients with pre-existing cardiovascular diseases (CVDs) have a higher risk for severe illness, cardiac damage, and death. Therefore, strategies for cardiovascular protection in COVID-19 are urgently needed until a reliable and effective treatment for COVID-19 (e.g., vaccine) is established. Because angiotensin-converting enzyme 2 (ACE2) is overexpressed in the cardiovascular system during CVD, COVID-19 patients with pre-existing CVDs may have higher risk for direct SARS-CoV-2 infection into the heart. However, the specific molecular mechanisms linking SARS-CoV-2 and cardiac risk are still unclear. Previous studies for SARS suggest that several SARS-CoV-1 genes [Envelope (E) protein, open reading frame (ORF) 3a, and ORF8a] have the potential to form viral channels (i.e., “viroporins”) in host cells and dysregulate host-cell functions. Hypothesis: SARS-CoV-2 genes encode viroporins, which can be expressed in the heart after infection and dysregulate cardiac electrophysiology and contractility. Methods: Individual SARS-CoV-2 genes were expressed in HEK293T cells via plasmids vector and the transfected cells were used for biochemical and electrophysiological assays. Results: By database analysis, we found that ORF3a and E from SARS-CoV-2 share over 85% similarity to those in SARS-CoV and maintain the critical sequences for ion channel functions. Both SARS-CoV-2-ORF3a and E protein form K + channels with similar channel properties of the endogenous transient outward K + current ( I to ), which can be activated during the initial repolarization phase of an action potential (AP) in cardiomyocytes. A computational model of human ventricular myocytes (O'Hara-Virag-Varro-Rudy Model) with increased I to amplitude suggests that the expression of these viroporins may increase Ca 2+ transient (CaT) amplitude at the baseline, but decrease CaT in response to increased heart rate. Conclusion: SARS-CoV-2 genes encode K + channels, which may dysregulate AP and Ca 2+ handing in cardiomyocytes, thus promoting decreased cardiac contractility and increased susceptibility to arrhythmias. Targeting these viroporins may reduce the risk of sudden cardiac death and cardiac damage in COVID-19 patients with pre-existing CVDs.
BackgroundHigh-NaCl diet is a contributing factor for cardiac hypertrophy. The role of HSP22 as a protective protein during cardiac hypertrophy due to hypernatremia is unclear. Accordingly, this study aimed to establish a cellular hypernatremic H9C2 model and to compare the expression of HSP22 in Ca2+ homeostasis between a high-NaCl and angiotensin II-induced hypertrophic cellular H9C2 model.MethodsReal-time PCR was performed to compare the mRNA expression. Flow cytometry and confocal microscopy were used to analyze the cells.ResultsThe addition of 30 mM NaCl for 48 h was the most effective condition for the induction of hypertrophic H9C2 cells (termed the in vitro hypernatremic model). Cardiac cellular hypertrophy was induced with 30 mM NaCl and 1 µM angiotensin II for 48 h, without causing abnormal morphological changes or cytotoxicity of the culture conditions. HSP22 contains a similar domain to that found in the consensus sequences of the late embryogenesis abundant protein group 3 from Artemia. The expression of HSP22 gradually decreased in the in vitro hypernatremic model. In contrast to the in vitro hypernatremic model, HSP22 increased after exposure to angiotensin II for 48 h. Intracellular Ca2+ decreased in the angiotensin II model and further decreased in the in vitro hypernatremic model. Impaired intracellular Ca2+ homeostasis was more evident in the in vitro hypernatremic model.ConclusionThe results showed that NaCl significantly decreased HSP22. Decreased HSP22, due to the hypernatremic condition, affected the Ca2+ homeostasis in the H9C2 cells. Therefore, hypernatremia induces cellular hypertrophy via impaired Ca2+ homeostasis. The additional mechanisms of HSP22 need to be explored further.
Introduction Contact sites between the endoplasmic reticulum (ER) and mitochondria (i.e., mitochondria‐associated membranes: MAMs) have important roles for the exchange of lipids, Ca2+, and reactive oxygen species (ROS), and greatly influence mitochondrial bioenergetics and cell fate. Mitofusin 2 (Mfn2), a mitochondrial fusion protein, is critical for MAM formation by tethering two organelles together to initiate contact. Although several post‐translational modifications (PTMs) of Mfn2 have been identified, including serine/threonine phosphorylation and ubiquitination, it remains unclear whether the PTMs of Mfn2 regulate its tethering function. In addition, while basal tyrosine phosphorylation (P‐Tyr) of Mfn2 was reported from mass spectroscopy data, the signaling pathways that regulate P‐Tyr levels of Mfn2 are completely unknown. Objective To determine whether P‐Tyr of Mfn2 modulates MAM functions. Methods Biochemical (mitochondrial fractionation), cell biological (Foster resonance energy transfer [FRET] efficiency between the outer mitochondrial membrane (OMM)‐targeted cyan fluorescent protein and ER membrane‐targeted yellow fluorescent protein), and physiological (imaging of mitochondrial Ca2+ [mtCa2+], ROS, and membrane potential [Δψm] in live cells) assays were performed in HEK293T cells. Results Endogenous expression of several tyrosine kinases, including proto‐oncogene tyrosine protein kinase (Src), C‐Terminal Src Kinase (CSK), and proline‐rich tyrosine kinase 2 (Pyk2), was found in the cytosolic and mitochondrial fractions of HEK293T cells. Overexpression of these proteins increased P‐Tyr levels of Mfn2, as detected by a general P‐Tyr antibody. Next, we found that CSK knockdown by shRNA in HEK293T cells enhances the physical coupling between the OMM and ER membrane compared to control cells, as determined by biochemical and live‐cell FRET assays. We also found that CSK knockdown induces mild, but significant, Δψm depolarization and increases basal mitochondrial ROS levels, which were quantified by a Δψm‐sensitive dye TMRE, and a mitochondria‐targeted H2O2 biosensor mt‐RoGFP2‐Orp1, respectively. Lastly, we observed mtCa2+ uptake in response to ER Ca2+ release induced by Gq protein‐coupled receptor stimulation, using a mitochondria‐targeted Ca2+ biosensor mt‐RCamp1h. Importantly, CSK‐knockdown enhanced mtCa2+ uptake in cells compared to control despite the mild Δψm depolarization. Conclusion Mfn2 has potential to be phosphorylated by tyrosine kinases in situ. P‐Tyr levels of Mfn2 may modulate the physical coupling and Ca2+ transport between organelles, which promotes mtCa2+‐dependent Δψm depolarization and mitochondrial ROS generation. Support or Funding Information A part of this research was supported by American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.
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