Relaxin alters cardiac myofilament function through a PKC-dependent pathway

2009 ◽  
Vol 297 (1) ◽  
pp. H29-H36 ◽  
Author(s):  
Erynn E. Shaw ◽  
Philip Wood ◽  
Justyna Kulpa ◽  
Feng Hua Yang ◽  
Alastair J. Summerlee ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Troponin I ◽  
Troponin T ◽  
Immunoblot Analysis ◽  
Inotropic Effects ◽  
Myosin Binding Protein ◽  
Myofilament Function ◽  
Cardiac Myofilament ◽  
Intracellular Ca ◽  
Myofilament Activation

The pregnancy hormone relaxin (RLX) is a powerful cardiostimulatory peptide. Despite its well-characterized effects on the heart, the intracellular mechanisms responsible for RLX's positive inotropic effects are unknown. Cardiac myofilaments are the central contractile elements of the heart, and changes in the phosphorylation status of myofilament proteins are known to mediate changes in function. The first objective of this study was to determine whether RLX stimulates myofilament activation and alters the phosphorylation of one or more myofilament proteins. RLX works through a variety of intracellular signaling cascades in different tissue types. Protein kinases A (PKA) and C (PKC) are two common molecules implicated in RLX signaling and are known to affect myofilament function. Thus the second objective of this study was to determine whether RLX mediates its myocardial effects through PKA or PKC activation. Murine myocardium was treated with recombinant H2-RLX, and cardiac myofilaments were isolated. RLX increased cardiac myofilament force development at physiological levels of intracellular Ca2+ without altering myofilament ATP consumption. Myosin binding protein C, troponin T, and troponin I phosphorylation levels were increased with RLX treatment. Immunoblot analysis revealed an increase in myofilament-associated PKC-δ, decreases in PKC-α and -βII, but no effect on PKC-ε. Inhibition of PKC with chelerythrine chloride or PKC-δ with rottlerin prevented the RLX-dependent changes in myofilament function and protein phosphorylation. PKA antagonism with H-89 had no effect on the myofilament effects of RLX. This study is the first to show that RLX-dependent changes in myofilament-associated PKC alters myofilament activation in a manner consistent with its cardiostimulatory effects.

Cardiac myofilament regulation by protein phosphatase type 1α and CapZ

2008 ◽  
Vol 86 (1) ◽  
pp. 70-78 ◽  
Author(s):  
Fenghua Yang ◽  
David L. Aiello ◽  
W. Glen Pyle
Keyword(s):  
Protein Phosphatase ◽  
Troponin I ◽  
Troponin T ◽  
Immunoblot Analysis ◽  
Intracellular Signalling ◽  
General Role ◽  
Myosin Light Chain 2 ◽  
Cardiac Myofilament ◽  
Myofilament Activation ◽  
Protein Phosphatase Type

Myofilament regulation by protein kinases is well characterized, but relatively little is known about protein phosphatase control of myofilaments. Increased protein phosphatase type 1 (PP1) activity observed in failing hearts underscores the need for investigation of this intracellular signal, including the elements that regulate its activity. The Z-disc protein CapZ controls protein kinase C (PKC) regulation of cardiac myofilaments, but whether this effect is specific to PKC, or CapZ plays a general role in intracellular signalling, is not known. We sought to determine how the α isoform of PP1 (PP1α) regulates murine cardiac myofilaments and whether CapZ influences PP1α-dependent regulation of cardiac myofilaments. Immunoblot analysis showed PP1α binding to cardiac myofilaments. Exogenous PP1α increased myofilament Ca2+ sensitivity and maximal actomyosin Mg2+-ATPase activity while dephosphorylating myosin binding protein C, troponin T, troponin I, and myosin light chain 2. Extraction of CapZ decreased myofilament-associated PP1α and attenuated the effects of PP1α on myofilament activation. PP1α-dependent dephosphorylation of myofilament proteins was reduced with CapZ extraction, except for troponin I. Extracting CapZ after PP1α treatment allowed most of the PP1α-dependent effects on myofilament activation to remain, indicating that CapZ removal modestly desensitizes cardiac myofilaments to dephosphorylation. Our results demonstrate myofilament regulation by PP1α and support the concept that cardiac Z-discs are vital components in intracellular signalling.

Troponin I serines 43/45 and regulation of cardiac myofilament function

2002 ◽  
Vol 283 (3) ◽  
pp. H1215-H1224 ◽  
Author(s):  
W. Glen Pyle ◽  
Marius P. Sumandea ◽  
R. John Solaro ◽  
Pieter P. De Tombe
Keyword(s):  
Cardiac Troponin ◽  
Troponin I ◽  
Troponin T ◽  
Fiber Bundles ◽  
Maximum Tension ◽  
Myofilament Function ◽  
Cardiac Myofilament ◽  
Myofilament Activation ◽  
Tension Cost ◽  
Mgatpase Activity

We studied Ca2+ dependence of tension and actomyosin ATPase rate in detergent extracted fiber bundles isolated from transgenic mice (TG), in which cardiac troponin I (cTnI) serines 43 and 45 were mutated to alanines (cTnI S43A/S45A). Basal phosphorylation levels of cTnI were lower in TG than in wild-type (WT) mice, but phosphorylation of cardiac troponin T was increased. Compared with WT, TG fiber bundles showed a 13% decrease in maximum tension and a 20% increase in maximum MgATPase activity, yielding an increase in tension cost. Protein kinase C (PKC) activation with endothelin (ET) or phenylephrine plus propranolol (PP) before detergent extraction induced a decrease in maximum tension and MgATPase activity in WT fibers, whereas ET or PP increased maximum tension and stiffness in TG fibers. TG MgATPase activity was unchanged by ET but increased by PP. Measurement of protein phosphorylation revealed differential effects of agonists between WT and TG myofilaments and within the TG myofilaments. Our results demonstrate the importance of PKC-mediated phosphorylation of cTnI S43/S45 in the control of myofilament activation and cross-bridge cycling rate.

scholarly journals Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function

2018 ◽  
Vol 45 (6) ◽  
pp. 2174-2186 ◽  
Author(s):  
Ni Zhu ◽  
Bing Yi ◽  
Zhifu Guo ◽  
Guanxin Zhang ◽  
Shengdong Huang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Cardiac Troponin I ◽  
Site Directed Mutagenesis ◽  
Functional Assay ◽  
Protective Effects ◽  
Diabetic Mice ◽  
Myofilament Function ◽  
Cardiac Myofilament

Background/Aims: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI), a key component in regulating myofilament function in the heart. Methods: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. Results: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1), a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. Conclusion: Our results demonstrate that Pim-1 is a novel kinase that phosphorylates cTnI primarily at Ser23/24 and Ser150 in cardiomyocytes, which in turn may modulate myofilament function under a variety of physiological and pathophysiological conditions.

Abstract 69: Cardiac Fibrosis Induced by Mkk6-p38 Activation Alters Myocardial Stiffness and Myofilament Function

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Farid Moussavi-Harami ◽  
Maria V Razumova ◽  
Jordan M Klaiman ◽  
Darrian Bugg ◽  
Michael Regnier ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Fibrosis ◽  
Troponin I ◽  
Troponin T ◽  
Systolic Function ◽  
Calcium Sensitivity ◽  
Matrix Composition ◽  
Passive Stiffness ◽  
Myocardial Stiffness ◽  
Myofilament Function

Heart failure with preserved ejection fraction (HFpEF) accounts for at least half of the patients with heart failure, but to date there are no proven therapies for these patients. Cardiac fibrosis and increased collagen content plays an important role in the pathophysiology of HFpEF. Studies in human HFpEF patients have indicated increased cardiac fibrosis, myocardial stiffness and myofilament calcium sensitivity. However, it is unclear how and if cardiac fibrosis directly alters myofilament function. In this study we used a new model of cardiac fibrosis genetically induced by the constitutive activation of MKK6-p38 (MKK6-Tg) signaling specifically in resident cardiac fibroblasts to determine the effect of fibrosis on myofilament stiffness and function. The MKK6-Tg mice develop interstitial and perivascular fibrosis in the heart accompanied with severe diastolic dysfunction, although systolic function remains normal. We measured the passive stiffness and isometric contractile properties of demembranated papillary muscle preparations. Our results show an increase in passive stiffness in demembranated papillary muscles from MKK6-Tg mice compared to NTG littermates when stretched to 24% from sarcomere length of 2.0 μM. Passive tension at SL=2.3 was significantly increased in MKK6-Tg mice (28±4 vs. 13±3 mN/mm 2 , P<0.05) compared to NTG mice, which was ascribed to changes in Titin isoform as well as extracellular matrix composition. The MKK6-Tg mice also show an increase in Ca 2+ sensitivity (pCa 50 =5.72±0.05 vs. 5.56±0.02, P<0.05). This was associated with an increased rate of force redevelopment in MKK6-Tg mice compared to NTG littermates (k TR =35±8 vs. 22±3 s -1 ). Towards understanding the mechanism of these myofilament changes we performed phosphate affinity electrophoresis and saw decreased phosphorylation of both Troponin I and Troponin T in MKK6-Tg hearts. There was no change in troponin I phosphorylation level at Ser23/24, indicating role of none-PKA mediated phosphorylation in mechanical changes seen in this model.

Molecular mechanisms of cardiac myofilament activation: modulation by pH and a troponin T mutant R92Q

2002 ◽  
Vol 97 (7) ◽  
pp. 1-1 ◽  
Author(s):  
R. John Solaro ◽  
Jamie Varghese ◽  
A. J. Marian ◽  
Murali Chandra
Keyword(s):  
Molecular Mechanisms ◽  
Troponin T ◽  
Cardiac Myofilament ◽  
Myofilament Activation

scholarly journals Troponin I chimera analysis of the cardiac myofilament tension response to protein kinase A

2001 ◽  
Vol 280 (2) ◽  
pp. C324-C332 ◽  
Author(s):  
Margaret V. Westfall ◽  
Immanuel I. Turner ◽  
Faris P. Albayya ◽  
Joseph M. Metzger
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Troponin I ◽  
Terminal Region ◽  
Adult Rat ◽  
Amino Terminal ◽  
Tension Response ◽  
Myosin Binding Protein ◽  
Cardiac Myofilament ◽  
Kinase A

Viral-mediated gene transfer of troponin I (TnI) isoforms and chimeras into adult rat cardiac myocytes was used to investigate the role TnI domains play in the myofilament tension response to protein kinase A (PKA). In myocytes expressing endogenous cardiac TnI (cTnI), PKA phosphorylated TnI and myosin-binding protein C and decreased the Ca2+ sensitivity of myofilament tension. In marked contrast, PKA did not influence Ca2+-activated tension in myocytes expressing the slow skeletal isoform of TnI or a chimera (N-slow/card-C TnI), which lack the unique phosphorylatable amino terminal extension found in cTnI. PKA-mediated phosphorylation of a second TnI chimera, N-card/slow-C TnI, which has the amino terminal region of cTnI, caused a decrease in the Ca2+ sensitivity of tension comparable in magnitude to control myocytes. Based on these results, we propose the amino terminal region shared by cTnI and N-card/slow-C TnI plays a central role in determining the magnitude of the PKA-mediated shift in myofilament Ca2+ sensitivity, independent of the isoform-specific functional domains previously defined within the carboxyl terminal backbone of TnI. Interestingly, exposure of permeabilized myocytes to acidic pH after PKA-mediated phosphorylation of cTnI resulted in an additive decrease in myofilament Ca2+ sensitivity. The isoform-specific, pH-sensitive region within TnI lies in the carboxyl terminus of TnI, and the additive response provides further evidence for the presence of a separate domain that directly transduces the PKA phosphorylation signal.

Differential effects of ovariectomy on calcium activation of cardiac and soleus myofilaments

1999 ◽  
Vol 277 (2) ◽  
pp. H467-H473 ◽  
Author(s):  
Jonggonnee Wattanapermpool ◽  
Peter J. Reiser
Keyword(s):  
Thin Filament ◽  
Troponin I ◽  
Left Ventricular ◽  
Hormone Deficiency ◽  
Ovariectomized Rats ◽  
Maximal Tension ◽  
Cardiac Myofilament ◽  
Myofilament Activation ◽  
Thin Filament Proteins ◽  
Ventricular Trabeculae

The hypothesis that ovarian sex hormone deficiency affects cardiac myofilament activation was tested. Chemically skinned ventricular trabeculae and single soleus muscle fibers were prepared from 10- and 14-wk ovariectomized and control rats. Tension-pCa (−log [Ca2+]) relations of left ventricular trabeculae and soleus fibers were compared to test whether thin filament proteins are potential sites of modulated activation. Trabeculae from ovariectomized rats exhibited a significant increase in Ca2+ sensitivity with no change in maximal tension-generating ability. In contrast, soleus fibers demonstrated no shift in Ca2+ sensitivity but generated significantly less maximal tension. No changes in thin filament protein isoform expression or loss of thin filament proteins were apparent in the trabeculae or soleus fibers from ovariectomized rats. Although not directly tested, our results are consistent with a possible modulation of regulatory proteins (e.g., cardiac troponin I) to account for the observed change in myofilament responsiveness of hearts from ovariectomized rats. Other possible mechanisms for the altered myocardial Ca2+ sensitivity after ovariectomy are discussed.

Transgenic incorporation of skeletal TnT into cardiac myofilaments blunts PKC-mediated depression of force

2001 ◽  
Vol 280 (3) ◽  
pp. H1011-H1018 ◽  
Author(s):  
David E. Montgomery ◽  
Murali Chandra ◽  
Qi-Quan Huang ◽  
Jian-Ping Jin ◽  
R. John Solaro
Keyword(s):  
Troponin I ◽  
Troponin T ◽  
Tension Development ◽  
Maximum Tension ◽  
Kinase C ◽  
Cardiac Myofilament ◽  
Skinned Fiber Bundles ◽  
The Impact ◽  
Pkc Activation

Protein kinase C (PKC)-mediated phosphorylation of cardiac troponin I (cTnI) and troponin T (cTnT) has been shown to diminish maximum activation of myofilaments. The functional role of cTnI phosphorylation has been investigated. However, the impact of cTnT phosphorylation on myofilament force is not well studied. We tested the effect of endogenous PKC activation on steady-state tension development and Ca2+ sensitivity in skinned fiber bundles from transgenic (TG) mouse hearts expressing fast skeletal TnT (fsTnT), which naturally lacks the PKC sites present in cTnT. The 12- O-tetradecanoylphorbol 13-acetate (TPA) treatment induced a 29% (46.1 ± 2.5 vs. 33.4 ± 2.6 mN/mm2) reduction in maximum tension in the nontransgenic (NTG) preparations ( n = 7) and was inhibited with chelerythrine. However, TPA did not induce a change in the maximum tension in the TG preparations ( n = 11). TPA induced a small but significant ( P < 0.02) increase in Ca2+sensitivity (untreated pCa50 = 5.63 ± 0.01 vs. treated pCa50 = 5.72 ± 0.01) only in TG preparations. In TG preparations, 32P incorporation was not evident in TnT and was also significantly diminished in cTnI, compared with NTG. Our data indicate that incorporation of fsTnT into the cardiac myofilament lattice blunts PKC-mediated depression of maximum tension. These data also suggest that cTnT may play an important role in amplifying the myofilament depression induced by PKC-mediated phosphorylation of cTnI.

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