Ranolazine Improves Cardiac Diastolic Dysfunction Through Modulation of Myofilament Calcium Sensitivity

Diabetes and heart failure are linked to NAD redox imbalance, whose role in diabetic cardiomyopathy has not been directly tested. Streptozotocin-induced diabetes in WT mice for 16 weeks promoted declines in systolic and diastolic function, which associated with lowered cardiac NAD/NADH ratio (NAD redox imbalance). To test the hypothesis that , we employed mouse models with cardiac-specific manipulations of NAD redox states. Cardiac-specific Ndufs4-KO mice (cKO) exhibit lowered cardiac NAD/NADH ratio with normal baseline function, geometry and energetics. Control and cKO mice were challenged with 8-week diabetic stress. Metabolomic analyses of plasma collected after the diabetic stress showed similar hyperglycemia and dyslipidemia stresses in diabetic control and diabetic cKO mice. Chronic diabetic stress promoted systolic and diastolic dysfunctions in control mice, which were further exacerbated in diabetic cKO mice in both male and female cohorts. Collagen levels and transcript analyses of fibrosis and extracellular matrix-dependent pathways showed no change in diabetic cKO hearts, suggesting that cardiomyocyte dysfunction is a likely culprit for the exacerbated dysfunction. Increased protein acetylation, including SOD2-K68Ac, was observed in diabetic cKO hearts. Inhibited antioxidant function by SOD2-K68Ac promoted protein oxidation in diabetic cKO hearts, suggesting oxidative stress as a pathogenic mechanism. We next examined phosphorylation status of myofilament proteins in these diabetic hearts. MyBPC-S282Pi levels are suppressed in failing hearts and remained unchanged in diabetic cKO hearts. TnI-S150Pi increases myofilament calcium sensitivity and prolongs calcium dissociation, while TnI-S23/24Pi imposes the opposite effects. TnI-S150Pi levels were elevated in diabetic cKO hearts, while TnI-S23/24Pi levels unchanged. Therefore, exacerbated diastolic dysfunction in diabetic cKO hearts is due to the selective phosphorylation at TnI-S150. AMPK is activated by energetic stress and phosphorylates TnI-S150. ATP levels decreased, and AMP/ATP ratio increased in diabetic cKO hearts, implicating impaired energetics to promote TnI-S150Pi and dysfunction. Elevation of NAD levels normalized cardiac NAD redox balance in diabetic cKO hearts. Elevated levels of SOD2-K68Ac and TnI-S150Pi, exacerbated systolic and diastolic dysfunction in diabetic cKO hearts were all reversed by elevation of NAD levels. Dysfunction in diabetic control hearts was also ameliorated by elevation of NAD levels. These data collectively conclude that NAD redox imbalance is a positive mediator of the progression of diabetic cardiomyopathy.

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PKC catalytic subunit and phenylephrine induce a comparable decrease in myofilament calcium sensitivity

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2007.03.129 ◽

2007 ◽

Vol 42 (6) ◽

pp. S46

Author(s):

Judit Barta ◽

Jolanda van der Velden ◽

Nicky M. Boontje ◽

Ruud Zaremba ◽

Ger J.M. Stienen

Keyword(s):

Calcium Sensitivity ◽

Catalytic Subunit ◽

Myofilament Calcium Sensitivity

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Abstract 219: Histidine Substituted Cardiac Troponin I (a164h) Improves Survival And Protects Cardiac Contractility During Acute Hypoxia In The Aged Murine Heart.

Circulation ◽

10.1161/circ.116.suppl_16.ii_23-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Nathan Palpant ◽

Sharlene Day ◽

Kimber Converso ◽

Joseph Metzger

Keyword(s):

Cardiac Troponin ◽

Troponin I ◽

Acute Hypoxia ◽

Cardiac Contractility ◽

Systolic Pressure ◽

Calcium Sensitivity ◽

The Elderly ◽

Cardiac Troponin I ◽

Pump Failure ◽

Myofilament Calcium Sensitivity

Contractile dysfunction associated with ischemia is a significant cause of morbidity and mortality particularly in the elderly. Strategies designed to protect the aged heart from ischemia-mediated pump failure are needed. We have generated transgenic (Tg) mice expressing a modified form of adult cardiac troponin I, the Ca ++ -activated molecular switch of the myofilament. Consonant with the fetal isoform, this transgene encodes a histidine substitution (A164H) in the critical switch domain of TnI thus increasing myofilament calcium sensitivity in a pH-dependent manner. We hypothesized that aged mice (24 months), intrinsically susceptible to myocardial dysfunction, would retain improved cardiac contractility at baseline and during an acute hypoxic challenge by means of myofilament-mediated calcium sensitization. Methods/Results: At baseline, by echocardiography, Tg hearts had increased systolic function, with a 26% higher mean ejection fraction compared to nontransgenic (Ntg) mice: 75 ± 3% [Tg: n = 13] vs. 63 ± 4% [Ntg: n = 12], P < 0.05, with no differences in diastolic function between the groups. To study the effects of acute hypoxia on cardiac hemodynamics mice underwent microconductance Millar catheterization while ventilated with 12% oxygen. Aged Tg mice had improved survival compared to Ntg mice: time to pump failure (65% of baseline peak systolic pressure) 11.59 ± 1.25 min. [Tg: n = 3] vs. 4.11 ± 1.90 min. [Ntg: n = 3], P < 0.05. After four minutes of hypoxia, Tg mice had markedly improved cardiac contractility compared to Ntg mice with increased stroke volume (30.05 ± 4.49 uL [Tg] vs. 13.23 ± 3.21 uL [Ntg], P < 0.05), end systolic pressure (106.09 ± 11.81 mmHg [Tg] vs. 64.49 ± 4.05 mmHg [Ntg], P < 0.05) and rate of positive left ventricular pressure development (12958.66 ± 2544.68 mmHg/sec [Tg] vs. 5717.00 ± 745.67 mmHg/sec [Ntg], P = 0.05). Conclusion: An alteration in myofilament calcium sensitivity via a pH-responsive histidine button in cardiac troponin I augments baseline heart function in Tg mice over their lifetime. During acute hypoxia, cTnI A164H improves survival in aged mice by maintaining cardiac contractility, and thus holds promise for the design of gene therapeutics to treat pump failure associated with acute ischemic events in the elderly.

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Abstract 223: Troponin I Tyrosine Phosphorylation Modulation of Cardiac Function

Circulation Research ◽

10.1161/res.121.suppl_1.223 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Elizabeth A Brundage ◽

Brendan Agatisa-Boyle ◽

Vikram Shettigar ◽

Jae-Hoon Chung ◽

Ziqing Qian ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Function ◽

Diastolic Dysfunction ◽

Troponin I ◽

Heart Function ◽

Calcium Sensitivity ◽

Regulatory Proteins ◽

Myocardial Relaxation ◽

Kinase Phosphorylation ◽

Accelerated Relaxation

Heart failure results in depressed contraction and slowed relaxation, both of which limit heart function and contribute to the progression of heart disease. Currently there is no chronic therapy to accelerate relaxation and reverse the diastolic dysfunction present in heart failure. Myocardial relaxation is regulated by serine/threonine phosphorylation of key regulatory proteins. Tyrosine (Tyr) specific kinases are expressed in the heart but the Tyr phosphorylation of regulatory proteins to modulate heart function has not been demonstrated. To investigate the effects of Tyr kinase phosphorylation on cardiac contraction we employed a novel cell penetrating peptide to deliver a direct Tyr kinase activator into isolated adult myocytes. Results demonstrate Tyr kinases activation increases Tyr phosphorylation of the regulatory protein troponin I (TnI) at Tyr26. We have demonstrated that TnI Tyr26 phosphorylation is beneficial to cardiac health by decreasing calcium sensitivity and accelerating myofilament deactivation (key determinants in accelerating myocardial relaxation) and that TnI Tyr26 phosphorylation undergoes functional integration with TnI Ser23/24 resulting in further accelerated calcium dissociation (accelerated relaxation) without further decreased calcium sensitivity (no further depression of contraction). We now demonstrate TnI Tyr26 also undergoes novel signaling integration with TnI Ser23/24 phosphorylation increasing the rate of Tyr kinase mediated Tyr26 phosphorylation. For the first time we demonstrate tyrosine kinase phosphorylation of TnI at Tyr26 modulates cardiac function resulting in accelerated relaxation. Increasing TnI Tyr26 phosphorylation may therefore serve as a novel targeted mechanism for future therapeutic development to accelerate depressed myocardial relaxation and improve diastolic dysfunction in heart failure.

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High-throughput assessment of calcium sensitivity in skinned cardiac myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.2.h969 ◽

2001 ◽

Vol 281 (2) ◽

pp. H969-H974 ◽

Cited By ~ 18

Author(s):

Chee Chew Lim ◽

Michiel H. B. Helmes ◽

Douglas B. Sawyer ◽

Mohit Jain ◽

Ronglih Liao

Keyword(s):

Cardiac Myocytes ◽

High Throughput ◽

High Throughput Screening ◽

Rapid Determination ◽

Isometric Force ◽

Calcium Sensitivity ◽

Pharmacological Agents ◽

Length Changes ◽

Myofilament Calcium Sensitivity ◽

High Degree

Isolated permeabilized cardiac myocytes have been used in the study of myofilament calcium sensitivity through measurement of the isometric force-pCa curve. Determining this force-pCa relationship in skinned myocytes is relatively expensive and carries a high degree of variability. We therefore attempted to establish an alternative high-throughput method to measure calcium sensitivity in cardiac myocytes. With the use of commercially available software that allows for precise measurement of sarcomere spacing, we measured sarcomere length changes in unloaded skinned cardiac myocytes over a range of calcium concentrations. With the use of this technique, we were able to accurately detect acute increases or decreases in myofilament calcium sensitivity after exposure to 10 mM caffeine or 5 mM 2,3-butanedione monoxime, respectively. This technique allows for the simple and rapid determination of myofilament calcium sensitivity in cardiac myocytes in a reproducible and inexpensive manner and could be used for high-throughput screening of pharmacological agents and/or transgenic mouse models for changes in myofilament calcium sensitivity.

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Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00134.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. H548-H556 ◽

Cited By ~ 6

Author(s):

Renan J. Sandoval ◽

Elisha R. Injeti ◽

James M. Williams ◽

William T. Georthoffer ◽

William J. Pearce

Keyword(s):

Calcium Influx ◽

Cerebral Arteries ◽

Calcium Sensitivity ◽

Cytosolic Calcium ◽

Stretch Ratio ◽

Myogenic Tone ◽

Postnatal Maturation ◽

Calcium Sensitization ◽

Myofilament Calcium Sensitivity ◽

Krebs Buffer

Regulation of cytosolic calcium and myofilament calcium sensitivity varies considerably with postnatal age in cerebral arteries. Because these mechanisms also govern myogenic tone, the present study used graded stretch to examine the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in term fetal compared with adult cerebral arteries. Term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses, with peak tensions averaging 24 and 26% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K+ (Kmax) at optimum stretch ratios (working diameter/unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic Ca2+ concentration at stretch ratios >2.0 in adult arteries, but increased Ca2+ concentration only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %Kmax values at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%) but were significantly less at pCa 8.0 (8.4 ± 2.3%). Conversely, adult %Kmax values at peak myogenic tone were significantly less at both pCa 8.0 (10.4 ± 1.8%) and pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced increases in myosin light chain phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The data demonstrate that the cerebrovascular myogenic response is highly conserved during postnatal maturation but is mediated differently in fetal and adult cerebral arteries.

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