Zhong Jian - Academia.edu (original) (raw)
Papers by Zhong Jian
Biophysical Journal, 2018
In VSMCs, Ca 2þ-activated Clchannels (CaCCs) are encoded by the gene TMEM16A/Anoctamin 1 (ANO1). ... more In VSMCs, Ca 2þ-activated Clchannels (CaCCs) are encoded by the gene TMEM16A/Anoctamin 1 (ANO1). The mechanism by which ANO1 influence the excitability of VSMCs remains to be elucidated due to questionable pharmacology and lack of a reliable genetic knockdown mouse model of ANO1. The aim of this study was to re-evaluate the role of ANO1 in electromechanical coupling of pulmonary artery (PA) smooth muscle using newer generation ANO1 blockers and a novel smooth muscle-specific inducible ANO1 knockout mouse model (SMC-iANO1-KO). Wire myography was used to determine the vascular reactivity to 5-HT of PA from wild-type and SMC-iANO1-KO mice. Calcium imaging experiments were also carried out using SMC-iGCaMP3 mice, which genetically express the Ca 2þ biosensor GCaMP3 in smooth muscle cells. 5-HT elicited a dose-dependent contraction (0.01-30 mM) that was similarly inhibited ($50-70%) by the ANO1 blocker CaCC Inh-A01 (10 mM), the Ca V 1.2 blocker nifedipine (1 mM) or the SERCA2 pump inhibitor cyclopiazonic acid (CPA; 10 mM). Genetic ablation of ANO1 produced a reduction in 5-HT-induced tone ($ 60% at 1 mM 5-HT) that was similar to that produced by CaCC inh A01, nifedipine or CPA. Ca 2þ imaging experiments in the intact PA of SMC-iGCaMP3 mice revealed that 5-HT evoked spatially and temporally localized Ca 2þ transients. These Ca 2þ oscillations were potently inhibited by CaCC Inh-A01 or nifedipine, and were abolished by CPA. In conclusion, 5-HT elicited highly localized Ca 2þ oscillations that were promoted by Ca 2þ entry through Ca V 1.2, most likely involving transient depolarizations evoked by ANO1 activated by a balance between oscillatory SR Ca 2þ release through IP 3 receptors and Ca 2þ entry through Ca V 1.2. We propose that the stable agonistinduced PA contraction results from the integration of stochastic and localized Ca 2þ events supported by a microenvironment comprising ANO1, Ca V 1.2 and IP 3 receptors.
Biophysical Journal, Feb 1, 2016
The diabetic patients, generally with overweight or obesity, face to heart diseases, while insuli... more The diabetic patients, generally with overweight or obesity, face to heart diseases, while insulin resistance is one of reasons that underlies abnormal cardiac electrophysiology. Insulin is a crucial hormone which regulates physiological processes such as energy metabolism, contractile activity, protein expression and ionic transports into/out of the cells. Besides, insulin provides glucose uptake to the cell via facilitating the localization of the glucose transporter, GLUT4, on the surface of the membrane. It has been proposed that this mechanism is deteriorated and therefore the electrical and mechanical activities of the heart are deteriorated on individuals who have insulin resistance and/or insulin deficiency. It is also proposed that insulin can activate different types of voltage-gated cation channels. We tested this hypothesis by studying the H9c2 cell line, derived from embryonic rat ventricular cells under both hyperglycemia and hyperinsulinemia. Basically, currents were recorded via whole cell configuration of the patch clamp technique. These cells were investigated on physiological, hyperglycemic (25-mM) and hyperglycemic (25-mM) plus hyperinsulinemic (100-nM) conditions. According to our findings on the cells, incubated 24-h with 25-mM glucose, both the peak values of inward and outward K þ-currents were significantly lower compare to the currents on the normo-glycemic condition. On the other hand, currents recorded after 24-hour of 25-mM glucose and 30-min of 100-nM insulin incubation (hyperglycemicþhyperinsulinemic) were significantly increased compare to hyperglycemic condition (p<0.05). Furhermore, 30 and 120-min of 100-nM insulin incubation to cells on normo-glycemic condition deteriorates K þ-currents and leads to hyperglycemia like effect. As a consequence, our data suggests that hyperglycemia-induced prolonged action potential in mammalian cells via reduction of the K þ-currents can underly the apperance of arrithymias in diabetic patients.
Proceedings of the National Academy of Sciences of the United States of America, Jul 29, 2021
Biophysical Journal, Feb 1, 2022
European Heart Journal, 2017
Vascular biology and the microcirculation 735 examined, the expression of 71 respective 34 genes ... more Vascular biology and the microcirculation 735 examined, the expression of 71 respective 34 genes was misregulated in CTEPH vs. HUVECs and CTEPH vs. HPAECs (False Discovery Rate: 0.05). Overall, markers of endothelial cells (VWF, CDH5, ICAM2) and apoptosis (CASP2, BAX, TP53, ANXA) were expressed on a similar level in all groups confirming the endothelial identity and non-apoptotic phenotype of cells outgrown from CTEPH tissue specimens. On the other hand, the signal intensity was most prominently misregulated in 14 pathways of biological processes. For example, proliferation markers (CCNA1, CCND1) were upregulated in CTEPH cells compared to HUVECs or HPAECs. Moreover, CTEPH endothelial cells exhibited a "profibrotic", "pro-coagulable" and "pro-inflammatory" phenotype, as suggested by the upregulation of genes involved in extracellular matrix production (
International Journal of Engineering Science, 2021
We develop a viscoelastic generalization of the elastic Eshelby inclusion solution, where the inc... more We develop a viscoelastic generalization of the elastic Eshelby inclusion solution, where the inclusion and surrounding matrix are two different viscoelastic solids and the inclusion's eigenstrain is a time-periodic oscillatory input. The solution exploits the Correspondence Principle of Linear Viscoelasticity and a Discrete Fourier Transform to efficiently capture the steady-state oscillatory behavior of the 3-D mechanical fields. The approach is illustrated here in the context of the recently-developed in vitro Cell-in-Gel system, where an isolated live cardiomyocyte (the inclusion) is paced to contract periodically within a soft hydrogel (the matrix), for the purpose of studying the effect of mechanical load on biochemical signals that regulate contractility. The addition of viscoelasticity improves the fidelity of our previous elastic Eshelby inclusion analysis of the Cell-in-Gel system by accounting for the time-varying fields and the resulting hysteresis and dissipated mechanical energy. This mathematical model is used to study the parametric sensitivities of the relative stiffness of the inclusion, the inclusion's aspect ratio (slenderness), and the cross-link density of the hydrogel matrix.
Journal of Molecular and Cellular Cardiology, 2017
Background: We embedded cardiomyocytes in a cross-linked hydrogel to study the effect of increase... more Background: We embedded cardiomyocytes in a cross-linked hydrogel to study the effect of increased afterload on in the heart. We observed that mechanically-loaded cardiomyocytes undergo noticeable changes in their Ca 2 + dynamics that can compensate for the increase in load. We have shown that these changes are mediated by the upregulation of nitric oxide (NO) signaling, which in turns affects Ca 2+ handling. Because many different Ca 2 + pathways could be affected we
Journal of Molecular and Cellular Cardiology, 2017
Author(s): Shimkunas, Rafael; Hegyi, Bence; Jian, Zhong; Coulibaly, Zana; Lam, Kit S; Ginsburg, K... more Author(s): Shimkunas, Rafael; Hegyi, Bence; Jian, Zhong; Coulibaly, Zana; Lam, Kit S; Ginsburg, Kenneth S; Bossuyt, Julie; Bers, Donald M; Izu, Leighton T; Chen-Izu, Ye
Biophysical Journal, 2018
Background: The heart must adjust its contractile force to compensate for the blood pressure chan... more Background: The heart must adjust its contractile force to compensate for the blood pressure changes in daily life (due to changes in posture, physical activity, emotional state, etc.) in order to maintain adequate cardiac output. Here we investigate the mechano-chemo-transduction (MCT) mechanisms that transduce mechanical load to regulate cardiac Ca 2þ signaling and contractility. Methods: We used a Cell-in-Gel system to embed freshly isolated rabbit ventricular myocytes in a 3-D viscoelastic hydrogel. The cell in-gel was continuously perfused with Tyrode's solution and electrically paced at 0.5 Hz. Selfcontrol experiments were conducted by (1) studying the myocytes contracting in-gel under mechanical load, (2) then dissolving the gel to release the cell into Tyrode's solution and (3) studying the cell contracting in solution load-free. Results: The cardiomyocytes contracting in-gel exhibited a larger cytosolic Ca 2þ transient than load-free cells (Fura-2 ratio 2.7150.07 vs 2.0850.04), revealing the MCT effect on regulating intracellular Ca 2þ. The SR Ca 2þ content was higher and the fractional release of Ca 2þ from SR was also larger when the cell was in-gel than load-free (measured with Fluo-5N), which should contribute to increasing the cytosolic Ca 2þ transient. Furthermore, the MCT effects on the SR Ca 2þ and cytosolic Ca 2þ were abolished by using 1 mM L-NAME to inhibit nitric oxide synthases (NOS), suggesting a critical role of NOS-NO signaling in mediating MCT. The cell in-gel contraction was reduced from 13.550.4% in untreated cell to 10.750.6% after L-NAME treatment, showing the MCT effect on enhancing contraction. Conclusion: Mechanochemo-transduction in cardiomyocyte is mediated by NOS-NO signaling to regulate the SR Ca 2þ release to increase the cytosolic Ca 2þ transient, which in turn enhances the contractility in compensatory response to increased load. This MCT mechanism contributes to the Anrep effect and is fundamental to cardiac regulation in health and disease.
Circulation Research, 2021
Biophysical Journal, 2018
Background: The heart must adjust its contractile force to compensate for the blood pressure chan... more Background: The heart must adjust its contractile force to compensate for the blood pressure changes in daily life (due to changes in posture, physical activity, emotional state, etc.) in order to maintain adequate cardiac output. Here we investigate the mechano-chemo-transduction (MCT) mechanisms that transduce mechanical load to regulate cardiac Ca 2þ signaling and contractility. Methods: We used a Cell-in-Gel system to embed freshly isolated rabbit ventricular myocytes in a 3-D viscoelastic hydrogel. The cell in-gel was continuously perfused with Tyrode's solution and electrically paced at 0.5 Hz. Selfcontrol experiments were conducted by (1) studying the myocytes contracting in-gel under mechanical load, (2) then dissolving the gel to release the cell into Tyrode's solution and (3) studying the cell contracting in solution load-free. Results: The cardiomyocytes contracting in-gel exhibited a larger cytosolic Ca 2þ transient than load-free cells (Fura-2 ratio 2.7150.07 vs 2.0850.04), revealing the MCT effect on regulating intracellular Ca 2þ. The SR Ca 2þ content was higher and the fractional release of Ca 2þ from SR was also larger when the cell was in-gel than load-free (measured with Fluo-5N), which should contribute to increasing the cytosolic Ca 2þ transient. Furthermore, the MCT effects on the SR Ca 2þ and cytosolic Ca 2þ were abolished by using 1 mM L-NAME to inhibit nitric oxide synthases (NOS), suggesting a critical role of NOS-NO signaling in mediating MCT. The cell in-gel contraction was reduced from 13.550.4% in untreated cell to 10.750.6% after L-NAME treatment, showing the MCT effect on enhancing contraction. Conclusion: Mechanochemo-transduction in cardiomyocyte is mediated by NOS-NO signaling to regulate the SR Ca 2þ release to increase the cytosolic Ca 2þ transient, which in turn enhances the contractility in compensatory response to increased load. This MCT mechanism contributes to the Anrep effect and is fundamental to cardiac regulation in health and disease.
Biophysical Journal, 2020
Biophysical Journal, 2019
Biophysical Journal, 2019
SPIE Proceedings, 2016
The objective of this study was to develop a method for simultaneously measuring the calcium and ... more The objective of this study was to develop a method for simultaneously measuring the calcium and contraction dynamics of single, live cardiomyocytes at high spatial resolutions. Such measurements are important to investigate local calcium release and the mechanical response at the sarcomere level (i.e. the basic unit of contraction), which have important implications in cardiac dysfunction and arrhythmias in conditions such as hypertension, atrial fibrillation, and myocardial infarction. Here, we describe a multimodal second harmonic generation (SHG) and two photon fluorescence (2PF) microscopy technique that is used to simultaneously measure subsarcomere calcium and contraction events at high spatial and temporal resolutions. The method takes advantage of the label-free nature of SHG for imaging the sarcomeres and the high spatial colocalization of the SHG signal and the fluorescence signal excited from calcium indicators. This microscope was used to measure calcium sparks and waves and associated contractions in subcellular microdomains, leading to the generation of subcellular strain. We anticipate this new imaging tool will play an important role in studying mechanical stress-induced heart disease.
Biophysical Journal, 2016
A mutation within the gamma 2 subunit (PRKAG2) of AMP protein kinase (AMPK) conferring constituti... more A mutation within the gamma 2 subunit (PRKAG2) of AMP protein kinase (AMPK) conferring constitutive activity results in a decreased heart rate (unpublished data); in contrast, mice null for the PRAKG2 gene have an increased heart rate and are unable to achieve a resting bradycardia following endurance training. Thus, it has become apparent that the gamma 2 subunit is essential for chronically reducing the heart rate at rest. Recently it has been demonstrated that endurance training induces alterations intrinsic to pacemaker cell function, where extensive classical evidence indicates that endurance training affects heart rate via increased vagal and decreased sympathetic tone. Autonomic influence on heart rate can be traced back to characteristic changes in time and frequency domain measures of heart rate variability. We tested the hypothesis that: (1) constitutively active AMPK, in addition to reducing the intrinsic heart rate, also modulates autonomic input to the heart, and (2) AMPK-dependent changes in autonomic activity are not only associated with characteristic changes in basal HRV, but also to variability intrinsic to sinoatrial nodal cells devoid of autonomic input. We show via telemetry, that unanesthetized mice homozygous for constitutively active AMPK display (1) a basal heart rate 50 BPM lower than wildtype littermates, (2) changes in rhythmicity, as noted by a 25% decrease in the coefficient of variation, (3) altered autonomic input, as seen with a two fold increase in very low frequency and corresponding decreases in both high and low frequency power, (4) an intrinsic heart rate approximately 65 BPM lower than control mice, and (5) a 30% increase in intrinsic variability. These findings provide the first in vivo evidence of AMPK's effects upon heart rate, heart rate variability, autonomic input in PRAKG2 associated mutations.
Biophysical Journal, 2016
characterized (Coppini et al. ABS Biophysical Journal 2015) the changes in sarcomere function and... more characterized (Coppini et al. ABS Biophysical Journal 2015) the changes in sarcomere function and E-C coupling that occur in ventricular myocardium of two HCM mouse models carrying different mutations in cTnT (R92Q and E163R). Both models exhibited diastolic dysfunction that was, however, related to different mechanisms i.e. E-C coupling abnormalities in R92Q and sarcomere changes in E163R. Here we employ these mouse models to study whether atrial remodeling is a consequence of diastolic dysfunction or is also influenced by the specific underlying mutation. Echocardiographic measurements of left atrial (LA) dimensions showed that LA area was severely increased in R92Q hearts while it was only mildly increased in E163R (in mm2 : 6.7350.5 in R92Q, 4.8250.16 in E163R vs 3.9750.26 in WT). Left atrial trabeculae were dissected and mounted isometrically to record twitch tension. We studied the steady-state force-frequency relationship and the response to positive inotropic stimuli such as Isoproterenol 10-7 mM (ISO) and 8 mM extracellular [Ca 2þ ]. Compared to WT, R92Q atrial trabeculae showed: (i) slower kinetics of both force development and relaxation (e.g. at 1 Hz, 50% relaxation was prolonged by 35%), (ii) impaired twitch amplitude at high pacing rates (50% reduction), (iii) depressed rested-state contractions and (iv) blunted increase of twitch tension in ISO and high [Ca 2þ ]. None of these changes were observed in intact E163R atrial trabeculae. These findings suggest that atrial remodeling in R92Q is more pronounced compared to E163R, and related to E-C coupling alterations. Supported by the Italian Ministry of Health (WFR GR-2011-02350583).
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](Biophysical Journal, 2016
Biophysical Journal, 2016
Ca 2þ signaling is central to cardiac excitation-contraction coupling and extensive studies have ... more Ca 2þ signaling is central to cardiac excitation-contraction coupling and extensive studies have revealed various Ca 2þ signaling events in ventricular myocytes. In systole, the action potential opens L-type Ca 2þ channels and triggers a synchronous release of Ca 2þ from SR, which causes a global Ca 2þ transient and whole cell contraction. During diastole, Ca 2þ is sequestered back into SR and the cytosolic Ca 2 concentration is kept low. However, pathological conditions can cause localized and spontaneous Ca 2þ release from SR seen as Ca 2þ sparks, puffs, embers, and waves. Spontaneous Ca 2þ waves can drive Na þ /Ca 2þ exchange current to depolarize the membrane potential, manifesting as delayed afterdepolarizations and triggered action potentials that are arrhythmogenic. Here we report a new type of Ca 2þ release event that is distinct from the previously known forms, named Ca 2þ tide. A Ca 2þ tide contains many discernable Ca 2þ sparks that occur near synchronously, but does not show a propagating wave front. Ca 2þ tides spontaneously arise after the systolic Ca 2þ transient in a contracting myocyte and can happen several times during diastole. Interestingly and importantly, the Ca 2þ tides are induced by mechanical loading of cardiomyocytes (by embedding cells in our Cell-in-Gel elastic matrix). Moreover, the cardiomyocytes isolated from TAC pressure-overload mouse model exhibited pronounced Ca 2þ tides under mechanical loading. In contrast, cardiomyocytes under loadfree conditions did not show any Ca 2þ tides. The mechanisms and conditions that give rise to the 'new kid on the block'-Ca 2þ tides-will be discussed.
Biophysical Journal, 2018
In VSMCs, Ca 2þ-activated Clchannels (CaCCs) are encoded by the gene TMEM16A/Anoctamin 1 (ANO1). ... more In VSMCs, Ca 2þ-activated Clchannels (CaCCs) are encoded by the gene TMEM16A/Anoctamin 1 (ANO1). The mechanism by which ANO1 influence the excitability of VSMCs remains to be elucidated due to questionable pharmacology and lack of a reliable genetic knockdown mouse model of ANO1. The aim of this study was to re-evaluate the role of ANO1 in electromechanical coupling of pulmonary artery (PA) smooth muscle using newer generation ANO1 blockers and a novel smooth muscle-specific inducible ANO1 knockout mouse model (SMC-iANO1-KO). Wire myography was used to determine the vascular reactivity to 5-HT of PA from wild-type and SMC-iANO1-KO mice. Calcium imaging experiments were also carried out using SMC-iGCaMP3 mice, which genetically express the Ca 2þ biosensor GCaMP3 in smooth muscle cells. 5-HT elicited a dose-dependent contraction (0.01-30 mM) that was similarly inhibited ($50-70%) by the ANO1 blocker CaCC Inh-A01 (10 mM), the Ca V 1.2 blocker nifedipine (1 mM) or the SERCA2 pump inhibitor cyclopiazonic acid (CPA; 10 mM). Genetic ablation of ANO1 produced a reduction in 5-HT-induced tone ($ 60% at 1 mM 5-HT) that was similar to that produced by CaCC inh A01, nifedipine or CPA. Ca 2þ imaging experiments in the intact PA of SMC-iGCaMP3 mice revealed that 5-HT evoked spatially and temporally localized Ca 2þ transients. These Ca 2þ oscillations were potently inhibited by CaCC Inh-A01 or nifedipine, and were abolished by CPA. In conclusion, 5-HT elicited highly localized Ca 2þ oscillations that were promoted by Ca 2þ entry through Ca V 1.2, most likely involving transient depolarizations evoked by ANO1 activated by a balance between oscillatory SR Ca 2þ release through IP 3 receptors and Ca 2þ entry through Ca V 1.2. We propose that the stable agonistinduced PA contraction results from the integration of stochastic and localized Ca 2þ events supported by a microenvironment comprising ANO1, Ca V 1.2 and IP 3 receptors.
Biophysical Journal, Feb 1, 2016
The diabetic patients, generally with overweight or obesity, face to heart diseases, while insuli... more The diabetic patients, generally with overweight or obesity, face to heart diseases, while insulin resistance is one of reasons that underlies abnormal cardiac electrophysiology. Insulin is a crucial hormone which regulates physiological processes such as energy metabolism, contractile activity, protein expression and ionic transports into/out of the cells. Besides, insulin provides glucose uptake to the cell via facilitating the localization of the glucose transporter, GLUT4, on the surface of the membrane. It has been proposed that this mechanism is deteriorated and therefore the electrical and mechanical activities of the heart are deteriorated on individuals who have insulin resistance and/or insulin deficiency. It is also proposed that insulin can activate different types of voltage-gated cation channels. We tested this hypothesis by studying the H9c2 cell line, derived from embryonic rat ventricular cells under both hyperglycemia and hyperinsulinemia. Basically, currents were recorded via whole cell configuration of the patch clamp technique. These cells were investigated on physiological, hyperglycemic (25-mM) and hyperglycemic (25-mM) plus hyperinsulinemic (100-nM) conditions. According to our findings on the cells, incubated 24-h with 25-mM glucose, both the peak values of inward and outward K þ-currents were significantly lower compare to the currents on the normo-glycemic condition. On the other hand, currents recorded after 24-hour of 25-mM glucose and 30-min of 100-nM insulin incubation (hyperglycemicþhyperinsulinemic) were significantly increased compare to hyperglycemic condition (p<0.05). Furhermore, 30 and 120-min of 100-nM insulin incubation to cells on normo-glycemic condition deteriorates K þ-currents and leads to hyperglycemia like effect. As a consequence, our data suggests that hyperglycemia-induced prolonged action potential in mammalian cells via reduction of the K þ-currents can underly the apperance of arrithymias in diabetic patients.
Proceedings of the National Academy of Sciences of the United States of America, Jul 29, 2021
Biophysical Journal, Feb 1, 2022
European Heart Journal, 2017
Vascular biology and the microcirculation 735 examined, the expression of 71 respective 34 genes ... more Vascular biology and the microcirculation 735 examined, the expression of 71 respective 34 genes was misregulated in CTEPH vs. HUVECs and CTEPH vs. HPAECs (False Discovery Rate: 0.05). Overall, markers of endothelial cells (VWF, CDH5, ICAM2) and apoptosis (CASP2, BAX, TP53, ANXA) were expressed on a similar level in all groups confirming the endothelial identity and non-apoptotic phenotype of cells outgrown from CTEPH tissue specimens. On the other hand, the signal intensity was most prominently misregulated in 14 pathways of biological processes. For example, proliferation markers (CCNA1, CCND1) were upregulated in CTEPH cells compared to HUVECs or HPAECs. Moreover, CTEPH endothelial cells exhibited a "profibrotic", "pro-coagulable" and "pro-inflammatory" phenotype, as suggested by the upregulation of genes involved in extracellular matrix production (
International Journal of Engineering Science, 2021
We develop a viscoelastic generalization of the elastic Eshelby inclusion solution, where the inc... more We develop a viscoelastic generalization of the elastic Eshelby inclusion solution, where the inclusion and surrounding matrix are two different viscoelastic solids and the inclusion's eigenstrain is a time-periodic oscillatory input. The solution exploits the Correspondence Principle of Linear Viscoelasticity and a Discrete Fourier Transform to efficiently capture the steady-state oscillatory behavior of the 3-D mechanical fields. The approach is illustrated here in the context of the recently-developed in vitro Cell-in-Gel system, where an isolated live cardiomyocyte (the inclusion) is paced to contract periodically within a soft hydrogel (the matrix), for the purpose of studying the effect of mechanical load on biochemical signals that regulate contractility. The addition of viscoelasticity improves the fidelity of our previous elastic Eshelby inclusion analysis of the Cell-in-Gel system by accounting for the time-varying fields and the resulting hysteresis and dissipated mechanical energy. This mathematical model is used to study the parametric sensitivities of the relative stiffness of the inclusion, the inclusion's aspect ratio (slenderness), and the cross-link density of the hydrogel matrix.
Journal of Molecular and Cellular Cardiology, 2017
Background: We embedded cardiomyocytes in a cross-linked hydrogel to study the effect of increase... more Background: We embedded cardiomyocytes in a cross-linked hydrogel to study the effect of increased afterload on in the heart. We observed that mechanically-loaded cardiomyocytes undergo noticeable changes in their Ca 2 + dynamics that can compensate for the increase in load. We have shown that these changes are mediated by the upregulation of nitric oxide (NO) signaling, which in turns affects Ca 2+ handling. Because many different Ca 2 + pathways could be affected we
Journal of Molecular and Cellular Cardiology, 2017
Author(s): Shimkunas, Rafael; Hegyi, Bence; Jian, Zhong; Coulibaly, Zana; Lam, Kit S; Ginsburg, K... more Author(s): Shimkunas, Rafael; Hegyi, Bence; Jian, Zhong; Coulibaly, Zana; Lam, Kit S; Ginsburg, Kenneth S; Bossuyt, Julie; Bers, Donald M; Izu, Leighton T; Chen-Izu, Ye
Biophysical Journal, 2018
Background: The heart must adjust its contractile force to compensate for the blood pressure chan... more Background: The heart must adjust its contractile force to compensate for the blood pressure changes in daily life (due to changes in posture, physical activity, emotional state, etc.) in order to maintain adequate cardiac output. Here we investigate the mechano-chemo-transduction (MCT) mechanisms that transduce mechanical load to regulate cardiac Ca 2þ signaling and contractility. Methods: We used a Cell-in-Gel system to embed freshly isolated rabbit ventricular myocytes in a 3-D viscoelastic hydrogel. The cell in-gel was continuously perfused with Tyrode's solution and electrically paced at 0.5 Hz. Selfcontrol experiments were conducted by (1) studying the myocytes contracting in-gel under mechanical load, (2) then dissolving the gel to release the cell into Tyrode's solution and (3) studying the cell contracting in solution load-free. Results: The cardiomyocytes contracting in-gel exhibited a larger cytosolic Ca 2þ transient than load-free cells (Fura-2 ratio 2.7150.07 vs 2.0850.04), revealing the MCT effect on regulating intracellular Ca 2þ. The SR Ca 2þ content was higher and the fractional release of Ca 2þ from SR was also larger when the cell was in-gel than load-free (measured with Fluo-5N), which should contribute to increasing the cytosolic Ca 2þ transient. Furthermore, the MCT effects on the SR Ca 2þ and cytosolic Ca 2þ were abolished by using 1 mM L-NAME to inhibit nitric oxide synthases (NOS), suggesting a critical role of NOS-NO signaling in mediating MCT. The cell in-gel contraction was reduced from 13.550.4% in untreated cell to 10.750.6% after L-NAME treatment, showing the MCT effect on enhancing contraction. Conclusion: Mechanochemo-transduction in cardiomyocyte is mediated by NOS-NO signaling to regulate the SR Ca 2þ release to increase the cytosolic Ca 2þ transient, which in turn enhances the contractility in compensatory response to increased load. This MCT mechanism contributes to the Anrep effect and is fundamental to cardiac regulation in health and disease.
Circulation Research, 2021
Biophysical Journal, 2018
Background: The heart must adjust its contractile force to compensate for the blood pressure chan... more Background: The heart must adjust its contractile force to compensate for the blood pressure changes in daily life (due to changes in posture, physical activity, emotional state, etc.) in order to maintain adequate cardiac output. Here we investigate the mechano-chemo-transduction (MCT) mechanisms that transduce mechanical load to regulate cardiac Ca 2þ signaling and contractility. Methods: We used a Cell-in-Gel system to embed freshly isolated rabbit ventricular myocytes in a 3-D viscoelastic hydrogel. The cell in-gel was continuously perfused with Tyrode's solution and electrically paced at 0.5 Hz. Selfcontrol experiments were conducted by (1) studying the myocytes contracting in-gel under mechanical load, (2) then dissolving the gel to release the cell into Tyrode's solution and (3) studying the cell contracting in solution load-free. Results: The cardiomyocytes contracting in-gel exhibited a larger cytosolic Ca 2þ transient than load-free cells (Fura-2 ratio 2.7150.07 vs 2.0850.04), revealing the MCT effect on regulating intracellular Ca 2þ. The SR Ca 2þ content was higher and the fractional release of Ca 2þ from SR was also larger when the cell was in-gel than load-free (measured with Fluo-5N), which should contribute to increasing the cytosolic Ca 2þ transient. Furthermore, the MCT effects on the SR Ca 2þ and cytosolic Ca 2þ were abolished by using 1 mM L-NAME to inhibit nitric oxide synthases (NOS), suggesting a critical role of NOS-NO signaling in mediating MCT. The cell in-gel contraction was reduced from 13.550.4% in untreated cell to 10.750.6% after L-NAME treatment, showing the MCT effect on enhancing contraction. Conclusion: Mechanochemo-transduction in cardiomyocyte is mediated by NOS-NO signaling to regulate the SR Ca 2þ release to increase the cytosolic Ca 2þ transient, which in turn enhances the contractility in compensatory response to increased load. This MCT mechanism contributes to the Anrep effect and is fundamental to cardiac regulation in health and disease.
Biophysical Journal, 2020
Biophysical Journal, 2019
Biophysical Journal, 2019
SPIE Proceedings, 2016
The objective of this study was to develop a method for simultaneously measuring the calcium and ... more The objective of this study was to develop a method for simultaneously measuring the calcium and contraction dynamics of single, live cardiomyocytes at high spatial resolutions. Such measurements are important to investigate local calcium release and the mechanical response at the sarcomere level (i.e. the basic unit of contraction), which have important implications in cardiac dysfunction and arrhythmias in conditions such as hypertension, atrial fibrillation, and myocardial infarction. Here, we describe a multimodal second harmonic generation (SHG) and two photon fluorescence (2PF) microscopy technique that is used to simultaneously measure subsarcomere calcium and contraction events at high spatial and temporal resolutions. The method takes advantage of the label-free nature of SHG for imaging the sarcomeres and the high spatial colocalization of the SHG signal and the fluorescence signal excited from calcium indicators. This microscope was used to measure calcium sparks and waves and associated contractions in subcellular microdomains, leading to the generation of subcellular strain. We anticipate this new imaging tool will play an important role in studying mechanical stress-induced heart disease.
Biophysical Journal, 2016
A mutation within the gamma 2 subunit (PRKAG2) of AMP protein kinase (AMPK) conferring constituti... more A mutation within the gamma 2 subunit (PRKAG2) of AMP protein kinase (AMPK) conferring constitutive activity results in a decreased heart rate (unpublished data); in contrast, mice null for the PRAKG2 gene have an increased heart rate and are unable to achieve a resting bradycardia following endurance training. Thus, it has become apparent that the gamma 2 subunit is essential for chronically reducing the heart rate at rest. Recently it has been demonstrated that endurance training induces alterations intrinsic to pacemaker cell function, where extensive classical evidence indicates that endurance training affects heart rate via increased vagal and decreased sympathetic tone. Autonomic influence on heart rate can be traced back to characteristic changes in time and frequency domain measures of heart rate variability. We tested the hypothesis that: (1) constitutively active AMPK, in addition to reducing the intrinsic heart rate, also modulates autonomic input to the heart, and (2) AMPK-dependent changes in autonomic activity are not only associated with characteristic changes in basal HRV, but also to variability intrinsic to sinoatrial nodal cells devoid of autonomic input. We show via telemetry, that unanesthetized mice homozygous for constitutively active AMPK display (1) a basal heart rate 50 BPM lower than wildtype littermates, (2) changes in rhythmicity, as noted by a 25% decrease in the coefficient of variation, (3) altered autonomic input, as seen with a two fold increase in very low frequency and corresponding decreases in both high and low frequency power, (4) an intrinsic heart rate approximately 65 BPM lower than control mice, and (5) a 30% increase in intrinsic variability. These findings provide the first in vivo evidence of AMPK's effects upon heart rate, heart rate variability, autonomic input in PRAKG2 associated mutations.
Biophysical Journal, 2016
characterized (Coppini et al. ABS Biophysical Journal 2015) the changes in sarcomere function and... more characterized (Coppini et al. ABS Biophysical Journal 2015) the changes in sarcomere function and E-C coupling that occur in ventricular myocardium of two HCM mouse models carrying different mutations in cTnT (R92Q and E163R). Both models exhibited diastolic dysfunction that was, however, related to different mechanisms i.e. E-C coupling abnormalities in R92Q and sarcomere changes in E163R. Here we employ these mouse models to study whether atrial remodeling is a consequence of diastolic dysfunction or is also influenced by the specific underlying mutation. Echocardiographic measurements of left atrial (LA) dimensions showed that LA area was severely increased in R92Q hearts while it was only mildly increased in E163R (in mm2 : 6.7350.5 in R92Q, 4.8250.16 in E163R vs 3.9750.26 in WT). Left atrial trabeculae were dissected and mounted isometrically to record twitch tension. We studied the steady-state force-frequency relationship and the response to positive inotropic stimuli such as Isoproterenol 10-7 mM (ISO) and 8 mM extracellular [Ca 2þ ]. Compared to WT, R92Q atrial trabeculae showed: (i) slower kinetics of both force development and relaxation (e.g. at 1 Hz, 50% relaxation was prolonged by 35%), (ii) impaired twitch amplitude at high pacing rates (50% reduction), (iii) depressed rested-state contractions and (iv) blunted increase of twitch tension in ISO and high [Ca 2þ ]. None of these changes were observed in intact E163R atrial trabeculae. These findings suggest that atrial remodeling in R92Q is more pronounced compared to E163R, and related to E-C coupling alterations. Supported by the Italian Ministry of Health (WFR GR-2011-02350583).
Biophysical Journal, 2016
Biophysical Journal, 2016
Ca 2þ signaling is central to cardiac excitation-contraction coupling and extensive studies have ... more Ca 2þ signaling is central to cardiac excitation-contraction coupling and extensive studies have revealed various Ca 2þ signaling events in ventricular myocytes. In systole, the action potential opens L-type Ca 2þ channels and triggers a synchronous release of Ca 2þ from SR, which causes a global Ca 2þ transient and whole cell contraction. During diastole, Ca 2þ is sequestered back into SR and the cytosolic Ca 2 concentration is kept low. However, pathological conditions can cause localized and spontaneous Ca 2þ release from SR seen as Ca 2þ sparks, puffs, embers, and waves. Spontaneous Ca 2þ waves can drive Na þ /Ca 2þ exchange current to depolarize the membrane potential, manifesting as delayed afterdepolarizations and triggered action potentials that are arrhythmogenic. Here we report a new type of Ca 2þ release event that is distinct from the previously known forms, named Ca 2þ tide. A Ca 2þ tide contains many discernable Ca 2þ sparks that occur near synchronously, but does not show a propagating wave front. Ca 2þ tides spontaneously arise after the systolic Ca 2þ transient in a contracting myocyte and can happen several times during diastole. Interestingly and importantly, the Ca 2þ tides are induced by mechanical loading of cardiomyocytes (by embedding cells in our Cell-in-Gel elastic matrix). Moreover, the cardiomyocytes isolated from TAC pressure-overload mouse model exhibited pronounced Ca 2þ tides under mechanical loading. In contrast, cardiomyocytes under loadfree conditions did not show any Ca 2þ tides. The mechanisms and conditions that give rise to the 'new kid on the block'-Ca 2þ tides-will be discussed.