Long-chain acylcarnitines (LCAC) are implicated in ischemia-reperfusion (I/R)-induced myocardial injury and mitochondrial dysfunction. Yet, molecular mechanisms underlying involvement of LCAC in cardiac injury are not sufficiently studied. It is known that in cardiomyocytes, palmitoylcarnitine (PC) can induce cytosolic Ca²⁺ accumulation, implicating L-type calcium channels, Na⁺/Ca²⁺ exchanger, and Ca²⁺-release from sarcoplasmic reticulum (SR). Alternatively, PC can evoke dissipation of mitochondrial potential (ΔΨ_m) and mitochondrial permeability transition pore (mPTP). Here, to dissect the complex nature of PC action on Ca²⁺ homeostasis and oxidative phosphorylation (OXPHOS) in cardiomyocytes and mitochondria, the methods of fluorescent microscopy, perforated path-clamp, and mitochondrial assays were used. We found that LCAC in dose-dependent manner can evoke Ca²⁺-sparks and oscillations, long-living Ca²⁺ enriched microdomains, and, finally, Ca²⁺ overload leading to hypercontracture and cardiomyocyte death. Collectively, PC-driven cardiotoxicity involves: (I) redistribution of Ca²⁺ from SR to mitochondria with minimal contribution of external calcium influx; (II) irreversible inhibition of Krebs cycle and OXPHOS underlying limited mitochondrial Ca²⁺ buffering; (III) induction of mPTP reinforced by PC-calcium interplay; (IV) activation of Ca²⁺-dependent phospholipases cPLA2 and PLC. Based on the inhibitory analysis we may suggest that simultaneous inhibition of both phospholipases could be an effective strategy for protection against PC-mediated toxicity in cardiomyocytes.

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca²⁺ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca²⁺ current (I_CaL). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of I_CaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of I_CaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting.

Cardiotoxins (CaTx) of the three-finger toxin family are one of the main components of cobra venoms. Depending on the structure of the N-terminal or the central polypeptide loop, they are classified into either group I and II or P- and S-types, respectively, and toxins of different groups or types interact with lipid membranes variably. While their main target in the organism is the cardiovascular system, there is no data on the effects of CaTxs from different groups or types on cardiomyocytes. To evaluate these effects, a fluorescence measurement of intracellular Ca²⁺ concentration and an assessment of the rat cardiomyocytes’ shape were used. The obtained results showed that CaTxs of group I containing two adjacent proline residues in the N-terminal loop were less toxic to cardiomyocytes than group II toxins and that CaTxs of S-type were less active than P-type ones. The highest activity was observed for Naja oxiana cobra cardiotoxin 2, which is of P-type and belongs to group II. For the first time, the effects of CaTxs of different groups and types on the cardiomyocytes were studied, and the data obtained showed that the CaTx toxicity to cardiomyocytes depends on the structures both of the N-terminal and central polypeptide loops.

—Aberrations in mechanisms of cardiac adaptation to catecholamine overflow can result in maladaptive cardiac hypertrophy and heart failure. Currently available heart failure therapies fall...

Various models, including stem cells derived and isolated cardiomyocytes with overexpressed channels, are utilized to analyze the functional interplay of diverse ion currents involved in cardiac automaticity and excitation–contraction coupling control. Here, we used β-NAD and ammonia, known hyperpolarizing and depolarizing agents, respectively, and applied inhibitory analysis to reveal the interplay of several ion channels implicated in rat papillary muscle contractility control. We demonstrated that: 4 mM β-NAD, having no strong impact on resting membrane potential (RMP) and action potential duration (APD90) of ventricular cardiomyocytes, evoked significant suppression of isometric force (F) of paced papillary muscle. Reactive blue 2 restored F to control values, suggesting the involvement of P2Y-receptor-dependent signaling in β-NAD effects. Meantime, 5 mM NH₄Cl did not show any effect on F of papillary muscle but resulted in significant RMP depolarization, APD90 shortening, and a rightward shift of I–V relationship for total steady state currents in cardiomyocytes. Paradoxically, NH₄Cl, being added after β-NAD and having no effect on RMP, APD, and I–V curve, recovered F to the control values, indicating β-NAD/ammonia antagonism. Blocking of HCN, Kir2.x, and L-type calcium channels, Ca²⁺-activated K⁺ channels (SK, IK, and BK), or NCX exchanger reverse mode prevented this effect, indicating consistent cooperation of all currents mediated by these channels and NCX. We suggest that the activation of Kir2.x and HCN channels by extracellular K⁺, that creates positive and negative feedback, and known ammonia and K⁺ resemblance, may provide conditions required for the activation of all the chain of channels involved in the interplay. Here, we present a mechanistic model describing an interplay of channels and second messengers, which may explain discovered antagonism of β-NAD and ammonia on rat papillary muscle contractile activity.

Snake venom phospholipase A2 (PLA2) affect, among others, the cardiovascular system; however, data on their effect on isolated cardiomyocytes are practically absent. In this work, we investigated the influence of HDP-1 and HDP-2, heterodimeric PLA2s from the venom of viper Vipera nikolskii, on isolated rat cardiomyocytes. Cardiomyocytes were stained with fluorescent probes and fluorescence was measured using Cell Observer fluorescent station. After PLA2 application, there was a prolonged lag phase with no changes in cell morphology or probe signal, followed by fluctuations in the cytosolic calcium level and cell hypercontracture with subsequent disruption of plasma membrane. In individual cardiomyocytes, time from the toxin addition until the hypercontracture varied by more than two-fold. In experiments with electrical stimulation, at HDP-2 application, a “pause” in contractions was observed, characterized by a decrease in the amplitude of the fluorescent response. Then, the contraction rhythm recovered, and later, hypercontracture was observed followed by cell destruction. Depending on times to these events, all cardiomyocytes studied were classified into two groups with fast and slow response to PLA2 action. The observed differences in the sensitivity of cardiomyocytes may be explained by unequal concentrations of intracellular calcium due to the different states of the cells at isolation.