Non-invasive biomarkers prognostic of decompensation events in NASH cirrhosis: a systematic literature review.

Liver cirrhosis due to nonalcoholic steatohepatitis (NASH) is a life-threatening condition with increasing incidence world-wide. Although its symptoms are unspecific, it can lead to decompensation events such as ascites, hepatic encephalopathy, variceal hemorrhage, and hepatocellular carcinoma (HCC). In addition, an increased risk for cardiovascular events has been demonstrated in patients with NASH. Pharmacological treatments for NASH cirrhosis are not yet available, one of the reasons being the lack in surrogate endpoints available in clinical trials of NASH cirrhosis. The feasibility of non-invasive prognostic biomarkers makes them interesting candidates as possible surrogate endpoints if their change following treatment would result in better outcomes for patients in future clinical trials of NASH cirrhosis. In this systematic literature review, a summary of the available literature on the prognostic performance of non-invasive biomarkers in terms of cardiovascular events, liver-related events, and mortality is outlined. Due to the scarcity of data specific for NASH cirrhosis, this review includes studies on NAFLD whose evaluation focuses on cirrhosis. Our search strategy identified the following non-invasive biomarkers with prognostic value in studies of NASH patients: NAFLD fibrosis score (NFS), Fibrosis-4 (FIB-4), aspartate aminotransferase (AST) to platelet ratio index (APRI), enhanced liver fibrosis (ELF™), BARD (BMI, AST/ALT (alanine aminotransferase) ratio, diabetes), Hepamet Fibrosis Score (HFS), liver enzymes (AST + ALT), alpha-fetoprotein, platelet count, neutrophil to lymphocyte ratio (NLR), Lysyl oxidase-like (LOXL) 2, miR-122, liver stiffness, MEFIB (liver stiffness measured with magnetic resonance elastography (MRE) + FIB-4), and PNPLA3 GG genotype. The aim of the present systematic literature review is to provide the reader with a summary of the non-invasive biomarkers with prognostic value in NASH cirrhosis and give an evaluation of their utility as treatment monitoring biomarkers in future clinical trials.

Unbiased kidney-centric molecular categorization of chronic kidney disease as a step towards precision medicine.

Current classification of chronic kidney disease (CKD) into stages using indirect systemic measures (estimated glomerular filtration rate (eGFR) and albuminuria) is agnostic to the heterogeneity of underlying molecular processes in the kidney thereby limiting precision medicine approaches. To generate a novel CKD categorization that directly reflects within kidney disease drivers we analyzed publicly available transcriptomic data from kidney biopsy tissue. A Self-Organizing Maps unsupervised artificial neural network machine-learning algorithm was used to stratify a total of 369 patients with CKD and 46 living kidney donors as healthy controls. Unbiased stratification of the discovery cohort resulted in identification of four novel molecular categories of disease termed CKD-Blue, CKD-Gold, CKD-Olive, CKD-Plum that were replicated in independent CKD and diabetic kidney disease datasets and can be further tested on any external data at kidneyclass.org. Each molecular category spanned across CKD stages and histopathological diagnoses and represented transcriptional activation of distinct biological pathways. Disease progression rates were highly significantly different between the molecular categories. CKD-Gold displayed rapid progression, with significant eGFR-adjusted Cox regression hazard ratio of 5.6 [1.01-31.3] for kidney failure and hazard ratio of 4.7 [1.3-16.5] for composite of kidney failure or a 40% or more eGFR decline. Urine proteomics revealed distinct patterns between the molecular categories, and a 25-protein signature was identified to distinguish CKD-Gold from other molecular categories. Thus, patient stratification based on kidney tissue omics offers a gateway to non-invasive biomarker-driven categorization and the potential for future clinical implementation, as a key step towards precision medicine in CKD.

Deletion of the C-terminal phosphorylation sites in the cardiac ß-subunit does not affect the basic ß-adrenergic response of the heart and the Ca(v)1.2 channel.

Phosphorylation of the cardiac ß subunit (Ca(v)ß(2)) of the Ca(v)1.2 L-type Ca(2+) channel complex has been proposed as a mechanism for regulation of L-type Ca(2+) channels by various protein kinases including PKA, CaMKII, Akt/PKB, and PKG. To test this hypothesis directly in vivo, we generated a knock-in mouse line with targeted mutation of the Ca(v)ß(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; ßStop mouse). This mutation prevented translation of the Ca(v)ß(2) C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac ßStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type I(Ca). The regulation of the L-type current by stimulation of the ß-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). ßStop mice were cross-bred with mice expressing the Ca(v)1.2 gene containing the mutation S1928A (SAßStop) or S1512A and S1570A (SFßStop) in the C terminus of the α(1C) subunit. The ß-adrenergic regulation of the cardiac I(Ca) was unaltered in these mouse lines. In contrast, truncation of the Ca(v)1.2 at Asp(1904) abolished ß-adrenergic up-regulation of I(Ca) in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Ca(v)ß(2), Ser(1928), Ser(1512), and Ser(1570) of the Ca(v)1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Ca(v)1.2 channel.

Cav1.2 L-type Ca²âº channels mediate cocaine-induced GluA1 trafficking in the nucleus accumbens, a long-term adaptation dependent on ventral tegmental area Ca(v)1.3 channels.

AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesoaccumbal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization, we have examined phosphorylation of GluA1 at key residues serine 845 (S845) and S831, as well as GluA1 cell surface levels in the nucleus accumbens (NAc) of cocaine-preexposed mice and the role of brain-specific Ca(v)1.2 and Ca(v)1.3 L-type Ca²âº channels (LTCCs), therein. We found higher basal levels of S845 phospho-GluA1 (P-GluA1) and cell surface GluA1 in the NAc following protracted withdrawal from cocaine exposure, changes that occur independently of LTCCs. In contrast, we found that a cocaine challenge that elicits expression of the cocaine-sensitized response increases S831 P-GluA1 that further increases surface GluA1 beyond the higher basal levels. Intra-NAc pharmacological manipulations indicate that the Ca(v)1.2-activated CaM kinase II (CaMKII) mediates cocaine-induced increase in S831 P-GluA1 and that both Ca(v)1.2-activated CaMKII and extracellular signal-regulated kinase 2 (ERK2) mediate the increase in GluA1 cell surface levels specific to the sensitized response. Experiments using adenoassociated viral vectors expressing Ca(v)1.3 and ERK2 siRNA further indicate that recruitment of the Ca(v)1.2 pathway in the NAc is dependent on ventral tegmental area Ca(v)1.3 LTCCs and ERK2. Together, these results identify candidate pathways that mediate cocaine-induced AMPAR plasticity in the NAc and provide a mechanism linking LTCCs and GluA1 plasticity to cocaine-induced persistent behavioral changes.

Facilitation and Ca2+-dependent inactivation are modified by mutation of the Ca(v)1.2 channel IQ motif.

The heart muscle responds to physiological needs with a short-term modulation of cardiac contractility. This process is determined mainly by properties of the cardiac L-type Ca(2+) channel (Ca(v)1.2), including facilitation and Ca(2+)-dependent inactivation (CDI). Both facilitation and CDI involve the interaction of calmodulin with the IQ motif of the Ca(v)1.2 channel, especially with Ile-1624. To verify this hypothesis, we created a mouse line in which Ile-1624 was mutated to Glu (Ca(v)1.2(I1624E) mice). Homozygous Ca(v)1.2(I1624E) mice were not viable. Therefore, we inactivated the floxed Ca(v)1.2 gene of heterozygous Ca(v)1.2(I1624E) mice by the α-myosin heavy chain-MerCreMer system. The resulting I/E mice were studied at day 10 after treatment with tamoxifen. Electrophysiological recordings in ventricular cardiomyocytes revealed a reduced Ca(v)1.2 current (I(Ca)) density in I/E mice. Steady-state inactivation and recovery from inactivation were modified in I/E versus control mice. In addition, voltage-dependent facilitation was almost abolished in I/E mice. The time course of I(Ca) inactivation in I/E mice was not influenced by the use of Ba(2+) as a charge carrier. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as a chelating agent for intracellular Ca(2+), inactivation of I(Ca) was slowed down in control but not I/E mice. The results show that the I/E mutation abolishes Ca(2+)/calmodulin-dependent regulation of Ca(v)1.2. The Ca(v)1.2(I1624E) mutation transforms the channel to a phenotype mimicking CDI.

Mouse and human induced pluripotent stem cells as a source for multipotent Isl1+ cardiovascular progenitors.

Ectopic expression of defined sets of genetic factors can reprogram somatic cells to create induced pluripotent stem (iPS) cells. The capacity to direct human iPS cells to specific differentiated lineages and to their progenitor populations can be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. During mouse cardiogenesis, the major lineages of the mature heart, cardiomyocytes, smooth muscle cells, and endothelial cells arise from a common, multipotent cardiovascular progenitor expressing the transcription factors Isl1 and Nkx2.5. Here we show, using genetic fate-mapping, that Isl1(+) multipotent cardiovascular progenitors can be generated from mouse iPS cells and spontaneously differentiate in all 3 cardiovascular lineages in vivo without teratoma. Moreover, we report the identification of human iPS-derived ISL1(+) progenitors with similar developmental potential. These results support the possibility to use patient-specific iPS-generated cardiovascular progenitors as a model to elucidate the pathogenesis of congenital and acquired forms of heart diseases.-Moretti, A., Bellin, M., Jung, C. B., Thies, T.-M., Takashima, Y., Bernshausen, A., Schiemann, M., Fischer, S., Moosmang, S., Smith, A. G., Lam, J. T., Laugwitz, K.-L. Mouse and human induced pluripotent stem cells as a source for multipotent Isl1(+) cardiovascular progenitors.

Unchanged beta-adrenergic stimulation of cardiac L-type calcium channels in Ca v 1.2 phosphorylation site S1928A mutant mice.

Phosphorylation of serine 1928 (Ser(1928)) of the cardiac Ca(v)1.2 subunit of L-type Ca(2+) channels has been proposed as the mechanism for regulation of L-type Ca(2+) channels by protein kinase A (PKA). To test this directly in vivo, we generated a knock-in mouse with targeted mutation of Ser(1928) to alanine. This mutation did not affect basal L-type current characteristics or regulation of the L-type current by PKA and the beta-adrenergic receptor, whereas the mutation abolished phosphorylation of Ca(v)1.2 by PKA. Therefore, our data show that PKA phosphorylation of Ser(1928) of Ca(v)1.2 is not functionally involved in beta-adrenergic stimulation of Ca(v)1.2-mediated Ca(2+) influx into the cardiomyocyte.

Calmodulin kinase II is involved in voltage-dependent facilitation of the L-type Cav1.2 calcium channel: Identification of the phosphorylation sites.

Calcium-dependent facilitation of L-type calcium channels has been reported to depend on the function of calmodulin kinase II. In contrast, the mechanism for voltage-dependent facilitation is not clear. In HEK 293 cells expressing Ca(v)1.2, Ca(v)beta2a, and calmodulin kinase II, the calcium current measured at +30 mV was facilitated up to 1.5-fold by a 200-ms-long prepulse to +160 mV. This voltage-dependent facilitation was prevented by the calmodulin kinase II inhibitors KN93 and the autocamtide-2-related peptide. In cells expressing the Ca(v)1.2 mutation I1649E, a residue critical for the binding of Ca2+-bound calmodulin, facilitation was also abolished. Calmodulin kinase II was coimmunoprecipitated with the Ca(v)1.2 channel from murine heart and HEK 293 cells expressing Ca(v)1.2 and calmodulinkinase II. The precipitated Ca(v)1.2 channel was phosphorylated in the presence of calmodulin and Ca2+. Fifteen putative calmodulin kinase II phosphorylation sites were identified mostly in the carboxyl-terminal tail of Ca(v)1.2. Neither truncation at amino acid 1728 nor changing the II-III loop serines 808 and 888 to alanines affected facilitation of the calcium current. In contrast, facilitation was decreased by the single mutations S1512A and S1570A and abolished by the double mutation S1512A/S1570A. These serines flank the carboxyl-terminal EF-hand motif. Immunoprecipitation of calmodulin kinase II with the Ca(v)1.2 channel was not affected by the mutation S1512A/S1570A. The phosphorylation of the Ca(v)1.2 protein was strongly decreased in the S1512A/S1570A double mutant. These results suggest that voltage-dependent facilitation of the Ca(v)1.2 channel depends on the phosphorylation of Ser1512/Ser1570 by calmodulin kinase II.