- Email: [email protected]
Transplantation of MSCs transfected with SHH gene ameliorates cardiac dysfunction after chronic myocardial infarction
Letters to the Editor
4997
Transplantation of MSCs transfected with SHH gene ameliorates cardiac dysfunction after chronic myocardial infarction Tao Tang, Ming Wu, Jinfu Yang ⁎ Department of the Cardiothoracic Surgery of the 2nd Xiangya Hospital, Central South University, Middle Renmin Road 139, 410011 Changsha, China
a r t i c l e
i n f o
Article history: Received 8 July 2013 Accepted 13 July 2013 Available online 1 August 2013 Keywords: SHH Cellular myoplasty Gene therapy Myocardial infarction
Dear Editor: Recently, it has been discovered that Sonic hedgehog (SHH) is important for the maintenance and repair of the adult cardiovascular system [1,2]. Thus, the SHH gene could be a possible candidate gene for gene therapy to ischemic heart disease. Kusano et al. injected naked DNA encoding the SHH gene into a myocardial infarction (MI) area and found that SHH gene transfer improved left ventricular function after MI [3]. At this stage, we have tried to transfer the SHH gene into mesenchymal stem cells (MSCs) and found that transplantation of MSCs transfected with SHH gene can ameliorate cardiac dysfunction after chronic MI. Firstly, we transferred the SHH gene into MSCs successfully. As the SHH precursor protein has the ability to autocatalyze, it can hence mature in cells [4]. Therefore the SHH-N segment was not selected as before, but instead the complete code sequence of SHH was used in our
study. The Nucleofector™ was used to transfer pmaxGFP into MSCs. The expression of green fluorescence was observed 24 h following transfection, which confirmed that the transfection efficiency was 30%–40%. 48 h following pcDNA3.1-SHH transfection, reverse transcription-PCR, Western blot analysis and real-time PCR demonstrated that the expression of SHH mRNA had significantly increased in transfected MSCs in comparison to untransfected MSCs. These results imply that MSCs cultured in vitro do not express SHH or express it at a low level, and that exogenous SHH gene transfer can be used to effectively express SHH in MSCs. The morphology of the transfected MSCs did not change, and both cytotoxicity and oncogenicity tests were negative. Secondly, we confirmed that the exogenous expression of the SHH gene can enhance angiogenesis through the SHH signaling pathway. Generally, it is thought that the level of Ptc 1, Gli and COUP-TFII expression can reflect the activity of the SHH signaling pathway [5]. In this study, we found that the expression of Ptc1, Gli-2 and COUP-TFII genes in the transfection group was significantly up-regulated 7 days after transplantation. This demonstrates that the exogenous expression of the SHH gene can also powerfully stimulate the SHH signaling pathway most dramatically. Following, real-time PCR evaluation further confirmed that the gene expression of angiogenic growth factors like VEGF and Ang-1 was up-regulated one week after transplantation, which was identical to a previously reported result [6]. Thus, we inferred that after the exogenous SHH gene was transfected into MSCs and the SHH protein was synthesized in cells, the active part of the SHH protein was secreted and released into the extracellular matrix exerting a paracrine effect. When SHH binds with the Ptc-1 receptor in the membrane of the mesenchymal cells, like fibroblasts, the downstream genes were activated, and the expression of
Fig. 1. The cardiac function of the intervention groups after chronic MI.
⁎ Corresponding author. E-mail address: [email protected] (J. Yang).
4998
Letters to the Editor
Fig. 2. Electron microscope examination. A: MSCs survived in MI areas (myocardial cells); B: the morphology of cell nuclei appeared spindle shaped; C: striated muscle fibers were observed in stem cells, demonstrating differentiation into cardiomyocytes; D: MSCs differentiated into cardiomyocytes.
angiogenic growth factors like VEGF and Ang-1 was promoted. Subsequently, angiogenesis is stimulated in local ischemic tissue coincident with the up-regulated expression of angiogenic growth factors. In our experiment, the capillary density of the transfection group was higher than that of other groups which confirmed that SHH gene therapy can promote the angiogenesis in a local MI area. Thirdly, we found that transplantation of MSCs transfected with SHH gene can ameliorate cardiac dysfunction after chronic myocardial infarction. Ventricular remodeling usually leads to cardiac function damage after MI. The results presented in our study confirm that the cardiac function of all groups was decreased after chronic MI. Compared to the control group, both the systolic function (LVSP and +dp/dtmax) and diastolic function (LVDP and −dp/dtmax) improved 4 and 8 weeks after the transplantation of MSCs transfected with the SHH gene Fig. 1. This demonstrated that the transplantation of MSCs transfected with the SHH gene has a therapeutic effect on chronic ischemic disease and can attenuate the persistent aggravation of cardiac function. There are two possible mechanisms of action. One is that the SHH gene induces the expression of angiogenic growth factors like VEGF and Ang-1 and thus promotes angiogenesis. Alternatively, MSCs differentiate into myocardial cells in the myocardium by the effect of “milieu-induceddifferentiation” and thus promotes myocardial regeneration. Our study showed that the transfected cells can survive and start differentiation in MI areas (See Fig. 2). In addition, SHH can also inhibit the apoptosis of myocardiac cells, attenuate myocardial fibrosis, and induce the migration of peripheral blood stem cells which may be helpful in improving cardiac function [6]. At the same time, this study also found that single MSC transplantation, SHH gene injection and mixed cell and gene injection can also improve the cardiac function following chronic MI, which corresponds with previous results [7,8]. It is worth noting that although the cardiac function of the transfection group was better than single MSC
transplantation, SHH gene injection, mixed cell and gene injection group, there was no statistical difference between these intervention groups. The reasons may be as follows: (1) Because a transient transfection technique was selected for gene transfection, the duration of SHH gene expression was limited to only about 1 week. This is supported by the fact that the expression of genes downstream of the SHH signaling pathway: Ptc1, Gli-2 and COUP-TFII and the angiogenic growth factors genes like VEGF and Ang-1, decreased at the second, fourth and eighth week, leading to a decrease in its effect of enhancing angiogenesis with time. (2) Electroporation did harm to the MSCs, and together with the cell loss observed during the early period of ischemic hypoxia in the MI area resulted in insufficient cell numbers which may also lead to the decrease of SHH gene expression. (3) The number of MSCs differentiating into myocardial cells in the myocardium by the effect of “milieu-induced-differentiation” is limited and decreases over time. This work was supported by the National Natural Science Foundation of China [grant number: 30871424].
References [1] Xiao Q, Hou N, Wang YP, et al. Impaired sonic hedgehog pathway contributes to cardiac dysfunction in type 1 diabetic mice with myocardial infarction. Cardiovasc Res Sep 1 2012;95(4):507–16. [2] Straface G, Aprahamian T, Flex A, et al. Sonic hedgehog regulates angiogenesis and myogenesis during post-natal skeletal muscle regeneration. J Cell Mol Med Aug 2009;13(8B):2424–35. [3] Kusano KF, Pola R, Murayama T, et al. Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling. Nat Med 2005;11(11):1197–204. [4] Zavros Y. The adventures of sonic hedgehog in development and repair. IV. Sonic hedgehog processing, secretion, and function in the stomach. Am J Physiol Gastrointest Liver Physiol May 2008;294(5):G1105–8. [5] Stanton BZ, Peng LF. Small-molecule modulators of the Sonic hedgehog signaling pathway. Mol Biosyst Jan 2010;6(1):44–54.
Letters to the Editor [6] Kusano KF, Pola R, Murayama T, et al. Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling. Nat Med Nov 2005;11(11):1197–204. [7] Schuleri KH, Feigenbaum GS, Centola M, et al. Autologous mesenchymal stem cells produce reverse remodelling in chronic ischaemic cardiomyopathy. Eur Heart J Nov 2009;30(22):2722–32.
4999
[8] Roncalli J, Renault MA, Tongers J, et al. Sonic hedgehog-induced functional recovery after myocardial infarction is enhanced by AMD3100-mediated progenitor-cell mobilization. J Am Coll Cardiol Jun 14 2011;57(24):2444–52.
0167-5273/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2013.07.126
Low-flow low-gradient aortic stenosis in patients with low ejection fraction: But is the flow truly low? Benoy Nalin Shah a,b,c, Navtej Singh Chahal a, Roxy Senior a,b,c,⁎ a b c
Department of Echocardiography, Royal Brompton Hospital, London, UK Cardiovascular Biomedical Research Unit, Royal Brompton Hospital, London, UK National Heart and Lung Institute, Imperial College, London, UK
a r t i c l e
i n f o
Article history: Received 8 July 2013 Accepted 13 July 2013 Available online 31 July 2013 Keywords: Aortic stenosis Low-flow low-gradient aortic stenosis Flow Ejection fraction
Dear Editor, It is known that judging the severity of aortic stenosis (AS) in patients with co-existent left ventricular (LV) systolic dysfunction can be challenging. These patients are particularly difficult for the managing clinician as their operative risk is high but outcome with medical therapy is poor. Quantitative Doppler echocardiography permits calculation of the aortic valve area (AVA), which may be b1.0 cm2 despite a low peak velocity and low mean pressure gradient. This widely accepted concept, first described over three decades ago [1], is termed low-flow low-gradient aortic stenosis (LFLG AS). In 1995, the use of dobutamine echocardiography (DbE) was first reported for helping to differentiate true severe AS from pseudo-severe AS [2], in which reduced systolic cusp excursion is secondary to depressed myocardial function. The theory underpinning the use of DbE was to augment systolic function and, thereby, permit reevaluation of Doppler parameters with an increased stroke volume. The current American [3] and European [4] guidelines on valvular heart disease both state that DbE may be helpful in the evaluation of patients with LFLG AS. The exact definition of LFLG AS has varied between studies, but has generally comprised the triad of an AVA b1.0 cm2, a low mean gradient (e.g. b40 mm Hg) [5] and a low ejection fraction (e.g. EF b 45%) [2]. Therefore, traditionally, it has been presumed that patients with significant LV dysfunction will – by definition – have low flow also. However, it has also been known for many years that the calculated AVA is directly related to trans-valvular flow [6]. At low trans-valvular flow-rates, the mean pressure gradient will be low irrespective of AVA ⁎ Corresponding author at: Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. Tel.: + 44 207 349 7740; fax: +44 207 351 8604. E-mail address: [email protected] (R. Senior).
though at a normal flow-rate, mean gradient reliably distinguishes between mild, moderate and severe AS [7]. Furthermore, in true severe AS (AVA b 1 cm2), once flow rate is normalized, the AVA does not change significantly with higher flow rates unlike with milder stenosis. The flow-rate across the aortic valve describes the volume of blood that is ejected during systole in 1 s and is thus easily calculated as the stroke volume (SV—in millilitres) divided by the ejection time (ET—in milliseconds), multiplied by 1000 (to achieve units of ml/s). Recently, in recognition of the heterogeneity of augmentation in flow rate achieved during DbE between patients, the concept of a projected valve area at a standardized flow-rate was proposed [8]. In patients with normal LV function, trans-aortic flow rates are around 250 ml/s, which is thus often considered as the cut-off value for a normal flow-rate [8]. However, both LV stroke volume and cardiac output (CO) can be normal despite significant LV systolic dysfunction. In a patient with cardiomyopathy, for example, end-diastolic (EDV) and end-systolic (ESV) volumes of 200 ml and 130 ml respectively yield a SV of 70 ml, even though EF is just 35%. If this individual has a resting heart rate of 70 bpm, the CO of 4.9 l/min is within normal parameters. Furthermore, flow-rate can also be normal in such an individual—if the patient had a long ET (e.g. 350 ms), the flow-rate would be low but with a shorter ET (e.g. 200 ms), the flow-rate would be normal. Additionally, a stroke volume of 70 ml at a heart rate of 70 bpm will generate a lower flow-rate compared to the same stroke volume at a heart rate of 80 bpm.
Table 1 Doppler echocardiographic data obtained at rest and following low-dose dobutamine infusion. Parameter
Rest
Low-dose dobutamine (5 μg/kg/min)
LVOT diameter: 23 mm LVOT VTI (cm) Stroke volume (ml) Ejection time (ms) Flow rate (ml/s) Aortic valve peak velocity (m/s) Aortic valve peak gradient (mm Hg) Aortic valve mean gradient (mm Hg) Aortic VTI (cm) Doppler velocity index Aortic valve area (cm2)
20.2 84 310 270 3.5 50 32 94.5 0.21 0.9
24.3 101 310 325 4.6 84 54 111.0 0.22 0.9
4997
Transplantation of MSCs transfected with SHH gene ameliorates cardiac dysfunction after chronic myocardial infarction Tao Tang, Ming Wu, Jinfu Yang ⁎ Department of the Cardiothoracic Surgery of the 2nd Xiangya Hospital, Central South University, Middle Renmin Road 139, 410011 Changsha, China
a r t i c l e
i n f o
Article history: Received 8 July 2013 Accepted 13 July 2013 Available online 1 August 2013 Keywords: SHH Cellular myoplasty Gene therapy Myocardial infarction
Dear Editor: Recently, it has been discovered that Sonic hedgehog (SHH) is important for the maintenance and repair of the adult cardiovascular system [1,2]. Thus, the SHH gene could be a possible candidate gene for gene therapy to ischemic heart disease. Kusano et al. injected naked DNA encoding the SHH gene into a myocardial infarction (MI) area and found that SHH gene transfer improved left ventricular function after MI [3]. At this stage, we have tried to transfer the SHH gene into mesenchymal stem cells (MSCs) and found that transplantation of MSCs transfected with SHH gene can ameliorate cardiac dysfunction after chronic MI. Firstly, we transferred the SHH gene into MSCs successfully. As the SHH precursor protein has the ability to autocatalyze, it can hence mature in cells [4]. Therefore the SHH-N segment was not selected as before, but instead the complete code sequence of SHH was used in our
study. The Nucleofector™ was used to transfer pmaxGFP into MSCs. The expression of green fluorescence was observed 24 h following transfection, which confirmed that the transfection efficiency was 30%–40%. 48 h following pcDNA3.1-SHH transfection, reverse transcription-PCR, Western blot analysis and real-time PCR demonstrated that the expression of SHH mRNA had significantly increased in transfected MSCs in comparison to untransfected MSCs. These results imply that MSCs cultured in vitro do not express SHH or express it at a low level, and that exogenous SHH gene transfer can be used to effectively express SHH in MSCs. The morphology of the transfected MSCs did not change, and both cytotoxicity and oncogenicity tests were negative. Secondly, we confirmed that the exogenous expression of the SHH gene can enhance angiogenesis through the SHH signaling pathway. Generally, it is thought that the level of Ptc 1, Gli and COUP-TFII expression can reflect the activity of the SHH signaling pathway [5]. In this study, we found that the expression of Ptc1, Gli-2 and COUP-TFII genes in the transfection group was significantly up-regulated 7 days after transplantation. This demonstrates that the exogenous expression of the SHH gene can also powerfully stimulate the SHH signaling pathway most dramatically. Following, real-time PCR evaluation further confirmed that the gene expression of angiogenic growth factors like VEGF and Ang-1 was up-regulated one week after transplantation, which was identical to a previously reported result [6]. Thus, we inferred that after the exogenous SHH gene was transfected into MSCs and the SHH protein was synthesized in cells, the active part of the SHH protein was secreted and released into the extracellular matrix exerting a paracrine effect. When SHH binds with the Ptc-1 receptor in the membrane of the mesenchymal cells, like fibroblasts, the downstream genes were activated, and the expression of
Fig. 1. The cardiac function of the intervention groups after chronic MI.
⁎ Corresponding author. E-mail address: [email protected] (J. Yang).
4998
Letters to the Editor
Fig. 2. Electron microscope examination. A: MSCs survived in MI areas (myocardial cells); B: the morphology of cell nuclei appeared spindle shaped; C: striated muscle fibers were observed in stem cells, demonstrating differentiation into cardiomyocytes; D: MSCs differentiated into cardiomyocytes.
angiogenic growth factors like VEGF and Ang-1 was promoted. Subsequently, angiogenesis is stimulated in local ischemic tissue coincident with the up-regulated expression of angiogenic growth factors. In our experiment, the capillary density of the transfection group was higher than that of other groups which confirmed that SHH gene therapy can promote the angiogenesis in a local MI area. Thirdly, we found that transplantation of MSCs transfected with SHH gene can ameliorate cardiac dysfunction after chronic myocardial infarction. Ventricular remodeling usually leads to cardiac function damage after MI. The results presented in our study confirm that the cardiac function of all groups was decreased after chronic MI. Compared to the control group, both the systolic function (LVSP and +dp/dtmax) and diastolic function (LVDP and −dp/dtmax) improved 4 and 8 weeks after the transplantation of MSCs transfected with the SHH gene Fig. 1. This demonstrated that the transplantation of MSCs transfected with the SHH gene has a therapeutic effect on chronic ischemic disease and can attenuate the persistent aggravation of cardiac function. There are two possible mechanisms of action. One is that the SHH gene induces the expression of angiogenic growth factors like VEGF and Ang-1 and thus promotes angiogenesis. Alternatively, MSCs differentiate into myocardial cells in the myocardium by the effect of “milieu-induceddifferentiation” and thus promotes myocardial regeneration. Our study showed that the transfected cells can survive and start differentiation in MI areas (See Fig. 2). In addition, SHH can also inhibit the apoptosis of myocardiac cells, attenuate myocardial fibrosis, and induce the migration of peripheral blood stem cells which may be helpful in improving cardiac function [6]. At the same time, this study also found that single MSC transplantation, SHH gene injection and mixed cell and gene injection can also improve the cardiac function following chronic MI, which corresponds with previous results [7,8]. It is worth noting that although the cardiac function of the transfection group was better than single MSC
transplantation, SHH gene injection, mixed cell and gene injection group, there was no statistical difference between these intervention groups. The reasons may be as follows: (1) Because a transient transfection technique was selected for gene transfection, the duration of SHH gene expression was limited to only about 1 week. This is supported by the fact that the expression of genes downstream of the SHH signaling pathway: Ptc1, Gli-2 and COUP-TFII and the angiogenic growth factors genes like VEGF and Ang-1, decreased at the second, fourth and eighth week, leading to a decrease in its effect of enhancing angiogenesis with time. (2) Electroporation did harm to the MSCs, and together with the cell loss observed during the early period of ischemic hypoxia in the MI area resulted in insufficient cell numbers which may also lead to the decrease of SHH gene expression. (3) The number of MSCs differentiating into myocardial cells in the myocardium by the effect of “milieu-induced-differentiation” is limited and decreases over time. This work was supported by the National Natural Science Foundation of China [grant number: 30871424].
References [1] Xiao Q, Hou N, Wang YP, et al. Impaired sonic hedgehog pathway contributes to cardiac dysfunction in type 1 diabetic mice with myocardial infarction. Cardiovasc Res Sep 1 2012;95(4):507–16. [2] Straface G, Aprahamian T, Flex A, et al. Sonic hedgehog regulates angiogenesis and myogenesis during post-natal skeletal muscle regeneration. J Cell Mol Med Aug 2009;13(8B):2424–35. [3] Kusano KF, Pola R, Murayama T, et al. Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling. Nat Med 2005;11(11):1197–204. [4] Zavros Y. The adventures of sonic hedgehog in development and repair. IV. Sonic hedgehog processing, secretion, and function in the stomach. Am J Physiol Gastrointest Liver Physiol May 2008;294(5):G1105–8. [5] Stanton BZ, Peng LF. Small-molecule modulators of the Sonic hedgehog signaling pathway. Mol Biosyst Jan 2010;6(1):44–54.
Letters to the Editor [6] Kusano KF, Pola R, Murayama T, et al. Sonic hedgehog myocardial gene therapy: tissue repair through transient reconstitution of embryonic signaling. Nat Med Nov 2005;11(11):1197–204. [7] Schuleri KH, Feigenbaum GS, Centola M, et al. Autologous mesenchymal stem cells produce reverse remodelling in chronic ischaemic cardiomyopathy. Eur Heart J Nov 2009;30(22):2722–32.
4999
[8] Roncalli J, Renault MA, Tongers J, et al. Sonic hedgehog-induced functional recovery after myocardial infarction is enhanced by AMD3100-mediated progenitor-cell mobilization. J Am Coll Cardiol Jun 14 2011;57(24):2444–52.
0167-5273/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2013.07.126
Low-flow low-gradient aortic stenosis in patients with low ejection fraction: But is the flow truly low? Benoy Nalin Shah a,b,c, Navtej Singh Chahal a, Roxy Senior a,b,c,⁎ a b c
Department of Echocardiography, Royal Brompton Hospital, London, UK Cardiovascular Biomedical Research Unit, Royal Brompton Hospital, London, UK National Heart and Lung Institute, Imperial College, London, UK
a r t i c l e
i n f o
Article history: Received 8 July 2013 Accepted 13 July 2013 Available online 31 July 2013 Keywords: Aortic stenosis Low-flow low-gradient aortic stenosis Flow Ejection fraction
Dear Editor, It is known that judging the severity of aortic stenosis (AS) in patients with co-existent left ventricular (LV) systolic dysfunction can be challenging. These patients are particularly difficult for the managing clinician as their operative risk is high but outcome with medical therapy is poor. Quantitative Doppler echocardiography permits calculation of the aortic valve area (AVA), which may be b1.0 cm2 despite a low peak velocity and low mean pressure gradient. This widely accepted concept, first described over three decades ago [1], is termed low-flow low-gradient aortic stenosis (LFLG AS). In 1995, the use of dobutamine echocardiography (DbE) was first reported for helping to differentiate true severe AS from pseudo-severe AS [2], in which reduced systolic cusp excursion is secondary to depressed myocardial function. The theory underpinning the use of DbE was to augment systolic function and, thereby, permit reevaluation of Doppler parameters with an increased stroke volume. The current American [3] and European [4] guidelines on valvular heart disease both state that DbE may be helpful in the evaluation of patients with LFLG AS. The exact definition of LFLG AS has varied between studies, but has generally comprised the triad of an AVA b1.0 cm2, a low mean gradient (e.g. b40 mm Hg) [5] and a low ejection fraction (e.g. EF b 45%) [2]. Therefore, traditionally, it has been presumed that patients with significant LV dysfunction will – by definition – have low flow also. However, it has also been known for many years that the calculated AVA is directly related to trans-valvular flow [6]. At low trans-valvular flow-rates, the mean pressure gradient will be low irrespective of AVA ⁎ Corresponding author at: Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. Tel.: + 44 207 349 7740; fax: +44 207 351 8604. E-mail address: [email protected] (R. Senior).
though at a normal flow-rate, mean gradient reliably distinguishes between mild, moderate and severe AS [7]. Furthermore, in true severe AS (AVA b 1 cm2), once flow rate is normalized, the AVA does not change significantly with higher flow rates unlike with milder stenosis. The flow-rate across the aortic valve describes the volume of blood that is ejected during systole in 1 s and is thus easily calculated as the stroke volume (SV—in millilitres) divided by the ejection time (ET—in milliseconds), multiplied by 1000 (to achieve units of ml/s). Recently, in recognition of the heterogeneity of augmentation in flow rate achieved during DbE between patients, the concept of a projected valve area at a standardized flow-rate was proposed [8]. In patients with normal LV function, trans-aortic flow rates are around 250 ml/s, which is thus often considered as the cut-off value for a normal flow-rate [8]. However, both LV stroke volume and cardiac output (CO) can be normal despite significant LV systolic dysfunction. In a patient with cardiomyopathy, for example, end-diastolic (EDV) and end-systolic (ESV) volumes of 200 ml and 130 ml respectively yield a SV of 70 ml, even though EF is just 35%. If this individual has a resting heart rate of 70 bpm, the CO of 4.9 l/min is within normal parameters. Furthermore, flow-rate can also be normal in such an individual—if the patient had a long ET (e.g. 350 ms), the flow-rate would be low but with a shorter ET (e.g. 200 ms), the flow-rate would be normal. Additionally, a stroke volume of 70 ml at a heart rate of 70 bpm will generate a lower flow-rate compared to the same stroke volume at a heart rate of 80 bpm.
Table 1 Doppler echocardiographic data obtained at rest and following low-dose dobutamine infusion. Parameter
Rest
Low-dose dobutamine (5 μg/kg/min)
LVOT diameter: 23 mm LVOT VTI (cm) Stroke volume (ml) Ejection time (ms) Flow rate (ml/s) Aortic valve peak velocity (m/s) Aortic valve peak gradient (mm Hg) Aortic valve mean gradient (mm Hg) Aortic VTI (cm) Doppler velocity index Aortic valve area (cm2)
20.2 84 310 270 3.5 50 32 94.5 0.21 0.9
24.3 101 310 325 4.6 84 54 111.0 0.22 0.9