Marius P. Sumandea

Functional consequences of caspase activation in cardiac myocytes

Cardiomyocyte apoptosis is present in many cardiac disease states, including heart failure and ischemic heart disease. Apoptosis is associated with the activation of caspases that mediate the cleavage of vital and structural proteins. However, the functional contribution of apoptosis to these conditions is not known. Furthermore, in cardiac myocytes, apoptosis may not be complete, allowing the cells to persist for a prolonged period within the myocardium. Therefore, we examined whether caspase-3 cleaved cardiac myofibrillar proteins and, if so, whether it affects contractile function. The effects of caspase-3 were studied in vitro on individual components of the cardiac myofilament including α-actin, α-actinin, myosin heavy chain, myosin light chain 1/2, tropomyosin, cardiac troponins (T, I, C), and the trimeric troponin complex. Exposure of the myofibrillar protein (listed above) to caspase-3 for 4 h resulted in the cleavage of α-actin and α-actinin, but not myosin heavy chain, myosin light chain 1/2, and tropomyosin, into three fragments (30, 20, and 15 kDa) and one major fragment (45 kDa), respectively. When cTnT, cTnI, and cTnC were incubated individually with caspase-3, there was no detectable cleavage. However, when the recombinant troponin complex was exposed to caspase-3, cTnT was cleaved, resulting in fragments of 25 kDa. Furthermore, rat cardiac myofilaments exposed to caspase-3 exhibited similar patterns of myofibrillar protein cleavage. Treatment with the caspase inhibitor DEVD-CHO or z-VAD-fmk abolished the cleavage. Myofilaments, isolated from adult rat ventricular myocytes after induction of apoptotic pathway by using β-adrenergic stimulation, displayed a similar pattern of actin and TnT cleavage. Exposure of skinned fiber to caspase-3 decreased maximal Ca2+-activated force and myofibrillar ATPase activity. Our results indicate that caspase-3 cleaved myofibrillar proteins, resulting in an impaired force/Ca2+ relationship and myofibrillar ATPase activity. Induction of apoptosis in cardiac cells was associated with similar cleavage of myofilaments. Therefore, activation of apoptotic pathways may lead to contractile dysfunction before cell death.

Augmented Protein Kinase C-α–Induced Myofilament Protein Phosphorylation Contributes to Myofilament Dysfunction in Experimental Congestive Heart Failure

It is becoming clear that upregulated protein kinase C (PKC) signaling plays a role in reduced ventricular myofilament contractility observed in congestive heart failure. However, data are scant regarding which PKC isozymes are involved. There is evidence that PKC-α may be of particular importance. Here, we examined PKC-α quantity, activity, and signaling to myofilaments in chronically remodeled myocytes obtained from rats in either early heart failure or end-stage congestive heart failure. Immunoblotting revealed that PKC-α expression and activation was unaltered in early heart failure but increased in end-stage congestive heart failure. Left ventricular myocytes were isolated by mechanical homogenization, Triton-skinned, and attached to micropipettes that projected from a force transducer and motor. Myofilament function was characterized by an active force–[Ca2+] relation to obtain Ca2+-saturated maximal force (Fmax) and myofilament Ca2+ sensitivity (indexed by EC50) before and after incubation with PKC-α, protein phosphatase type 1 (PP1), or PP2a. PKC-α treatment induced a 30% decline in Fmax and 55% increase in the EC50 in control cells but had no impact on myofilament function in failing cells. PP1-mediated dephosphorylation increased Fmax (15%) and decreased EC50 (≈20%) in failing myofilaments but had no effect in control cells. PP2a-dependent dephosphorylation had no effect on myofilament function in either group. Lastly, PP1 dephosphorylation restored myofilament function in control cells hyperphosphorylated with PKC-α. Collectively, our results suggest that in end-stage congestive heart failure, the myofilament proteins exist in a hyperphosphorylated state attributable, in part, to increased activity and signaling of PKC-α.

Augmented Phosphorylation of Cardiac Troponin I in Hypertensive Heart Failure

Background: Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility. Results: cTnI is hyperphosphorylated at Ser22/23 and Ser42/44 in spontaneously hypertensive rat of heart failure. Conclusion: The augmented phosphorylation of cTnI in hypertensive heart failure is correlated with elevated protein levels of PKC-α and -δ. Significance: This is the first in vivo evidence of cTnl phosphorylation at Ser42/44 in an animal model of hypertensive heart failure. An altered cardiac myofilament response to activating Ca2+ is a hallmark of human heart failure. Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility and Ca2+ sensitivity of cardiac muscle. cTnI can be phosphorylated by protein kinase A (PKA) at Ser22/23 and protein kinase C (PKC) at Ser22/23, Ser42/44, and Thr143. Whereas the functional significance of Ser22/23 phosphorylation is well understood, the role of other cTnI phosphorylation sites in the regulation of cardiac contractility remains a topic of intense debate, in part, due to the lack of evidence of in vivo phosphorylation. In this study, we utilized top-down high resolution mass spectrometry (MS) combined with immunoaffinity chromatography to determine quantitatively the cTnI phosphorylation changes in spontaneously hypertensive rat (SHR) model of hypertensive heart disease and failure. Our data indicate that cTnI is hyperphosphorylated in the failing SHR myocardium compared with age-matched normotensive Wistar-Kyoto rats. The top-down electron capture dissociation MS unambiguously localized augmented phosphorylation sites to Ser22/23 and Ser42/44 in SHR. Enhanced Ser22/23 phosphorylation was verified by immunoblotting with phospho-specific antibodies. Immunoblot analysis also revealed up-regulation of PKC-α and -δ, decreased PKCϵ, but no changes in PKA or PKC-β levels in the SHR myocardium. This provides direct evidence of in vivo phosphorylation of cTnI-Ser42/44 (PKC-specific) sites in an animal model of hypertensive heart failure, supporting the hypothesis that PKC phosphorylation of cTnI may be maladaptive and potentially associated with cardiac dysfunction.

Molecular and integrated biology of thin filament protein phosphorylation in heart muscle.

Abstract: An increasing body of evidence points to posttranslational modifications of the thin filament regulatory proteins, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) by protein kinase C (PKC) phosphorylation as important in both long‐ and short‐term regulation of cardiac function and potentially implicated in the transition between compensated hypertrophy and decompensation. The main sites for PKC‐dependent phosphorylation on cTnI are Ser43, Ser45, and Thr144 and on cTnT are Thr197, Ser201, Thr206, and Thr287 (mouse sequence). We analyzed the function of each phosphorylation residue using a phosphorylation mimic approach introducing glutamates (E) at PKC phosphorylation sites and then measuring the isometric tension of fiber bundles exchanged with these mutants. We also directly phosphorylated cTnI and cTnT by PKC, incorporated the phosphorylated troponins in the myofilament lattice, and determined the isometric tension at varying Ca2+ concentrations. We followed the experimental data with computational analysis prediction of helical content of cTnI and cTnT peptides that undergo phosphorylation. Here we summarize our recent data on the specific functional role of PKC phosphorylation sites of cTnI and cTnT.

Actin Capping Protein: An Essential Element in Protein Kinase Signaling to the Myofilaments

Actin capping protein (CapZ) binds the barbed ends of actin at sarcomeric Z-lines. In addition to anchoring actin, Z-discs bind protein kinase C (PKC). Although CapZ is crucial for myofibrillogenesis, its role in muscle function and intracellular signaling is unknown. We hypothesized that CapZ downregulation would impair myocardial function and disrupt PKC-myofilament signaling by impairing PKC–Z-disc interaction. To test these hypotheses, we examined transgenic (TG) mice in which cardiac CapZ protein is reduced. Fiber bundles were dissected from papillary muscles and detergent extracted. Some fiber bundles were treated with PKC activators phenylephrine (PHE) or endothelin (ET) before detergent extraction. We simultaneously measured Ca2+-dependent tension and actomyosin MgATPase activity. CapZ downregulation increased myofilament Ca2+ sensitivity without affecting maximum tension or actomyosin MgATPase activity. Maximum tension and actomyosin MgATPase activity were decreased after PHE or ET treatment of wild-type (WT) muscle. Fiber bundles from TG hearts did not respond to PHE or ET. Immunoblot analysis revealed an increase in myofilament-associated PKC-&egr; after PHE or ET exposure of WT preparations. In contrast, myofilament-associated PKC-&egr; was decreased after PHE or ET treatment in TG myocardium. Protein levels of myofilament-associated PKC-&bgr; were decreased in TG ventricle. C-protein and troponin I phosphorylation was increased after PHE or ET treatment in WT and TG hearts. Basal phosphorylation levels of C-protein and troponin I were higher in TG myocardium. These results indicate that downregulation of CapZ, or other changes associated with CapZ downregulation, increases cardiac myofilament Ca2+ sensitivity, inhibits PKC-mediated control of myofilament activation, and decreases myofilament-associated PKC-&bgr;.

Cardiac Troponin T, a Sarcomeric AKAP, Tethers Protein Kinase A at the Myofilaments

Efficient and specific phosphorylation of PKA substrates, elicited in response to β-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.

Altered signaling surrounding the C-lobe of cardiac troponin C in myofilaments containing an α-tropomyosin mutation linked to familial hypertrophic cardiomyopathy

A region of interaction between the near N-terminal of cardiac troponin I (cTnI) and the C-lobe of troponin C (cTnC), where troponin T (cTnT) binds, appears to be critical in regulation of myofilament Ca(2+)-activation. We probed whether functional consequences of modulation of this interface influence the function of tropomyosin (Tm) in thin filament activation. We modified the C-lobe of cTnC directly by addition of the Ca(2+)-sensitizer, EMD 57033, and indirectly by replacing native cTnI with cTnI-containing Glu residues at Ser-43 and Ser-45 (cTnI-S43E/S45E) in myofilaments from hearts of non-transgenic (NTG) and transgenic (TG) mice expressing a point mutation on alpha-Tm (E180G) linked to familial hypertrophic cardiomyopathy. Introduction of cTnI-S43E/S45E induced a significantly greater reduction in tension in TG myofilaments compared to NTG controls. Furthermore, the effect of EMD 57033 to restore Ca(2+)-sensitivity was higher in TG compared to NTG fiber bundles containing cTnI-S43E/S45E and compared to TG or NTG fiber bundles containing native TnI. Our results indicate that alterations in regions of interaction among the N-terminal of cTnI, the C-lobe of cTnC, and the C-terminus of cTnT are important in the regulation of myofilament activity. Although levels of phosphorylation at protein kinase C-dependent sites were the same in TG and NTG myofilaments, our data indicate that the effects of phosphorylation were more depressive in TG hearts.

Dissecting human skeletal muscle troponin proteoforms by top-down mass spectrometry

Skeletal muscles are the most abundant tissues in the human body. They are composed of a heterogeneous collection of muscle fibers that perform various functions. Skeletal muscle troponin (sTn) regulates skeletal muscle contraction and relaxation. sTn consists of 3 subunits, troponin I (TnI), troponin T (TnT), and troponin C (TnC). TnI inhibits the actomyosin Mg2+-ATPase, TnC binds Ca2+, and TnT is the tropomyosin (Tm)-binding subunit. The cardiac and skeletal isoforms of Tn share many similarities but the roles of modifications of Tn in the two muscles may differ. The modifications of cardiac Tn are known to alter muscle contractility and have been well-characterized. However, the modification status of sTn remains unclear. Here, we have employed top-down mass spectrometry (MS) to decipher the modifications of human sTnT and sTnI. We have extensively characterized sTnT and sTnI proteoforms, including alternatively spliced isoforms and post-translationally modified forms, found in human skeletal muscle with high mass accuracy and comprehensive sequence coverage. Moreover, we have localized the phosphorylation site of slow sTnT isoform III to Ser1 by tandem MS with electron capture dissociation. This is the first study to comprehensively characterize human sTn and also the first to identify the basal phosphorylation site for human sTnT by top-down MS.

Changes in Ca2+/Calmodulin-Dependent Protein Kinase (CaMKII) During Development of Hypertension-Induced Hypertrophy and Heart Failure

CaMKII is important in cardiac function and disease, and the two main cardiac isoforms (δC and δB) are targeted to cytosol and nucleus respectively. They regulate many ion channels and Ca2+ handling molecules in excitation-contraction coupling and also participate in transcriptional control and hypertrophic/ heart failure signaling. Here we investigate changes in δB and δC expression and activity during the development of Hypertensive Heart Diseases (HHD) through four progressive stages: (1) pre-hypertension, (2) onset of hypertension prior to hypertrophy, (3) overt hypertrophy, and (4) heart failure. Method: Western blots were used to measure the δB and δC expression and phosphorylation at Thr286/Thr305 (indicating enzyme activity) in the left ventricle of spontaneously hypertensive rat (SHR) at distinct stages of HHD vs. age-matched normotensive Wistar-Kyoto rat (WKY). MAJOR RESULTS: (1) δB and δC expression and phosphorylation in WKY gradually increased with age. (2) SHR show different δB and δC expression and phosphorylation from WKY, revealing disease-related changes apart from age-related changes. (3) Both δB and δC activity increased at the onset of hypertension, and persisted during hypertrophy and heart failure. (4) Interestingly, δB shows larger changes at earlier stages of hypertension and hypertrophy than δC, while δC shows larger changes at heart failure, implicating differential roles of each in regulating hypertrophy and transition to heart failure. (5) ACE inhibitor treatment of SHR at each disease stage effectively reduced hypertension and also reversed changes in δB and δC. CONCLUSION: CaMKII δB and δC are immediate responders to the onset of hypertension and mediate the development of hypertrophy and heart failure during chronic hypertension.